TY - JOUR
AU - Amer, Sawsan, M
AB - Abstract Nadifloxacin, mometasone furoate and miconazole nitrate are formulated together as a topical antifungal dosage form. In this work, a reversed-phase ultra-performance liquid chromatographic method coupled with a diode array detector (RP-UPLC-DAD) was developed and validated to determine nadifloxacin, mometasone furoate and miconazole nitrate simultaneously in their bulk powder, in pharmaceutical preparation and in spiked human plasma samples. Separation was achieved on an ACQUITY UPLC C18 column of 2.2 μm particle size (2.1 × 100 mm) via isocratic elution using a mobile phase consisting of methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate, then pH was adjusted to (7.00) using acetic acid, flow rate 0.6 mL/min, temperature 30°C and UV detection at 220 nm. The method is linear in a range from 5 to 400 μg/mL for both nadifloxacin and miconazole nitrate and from 20 to 500 μg/mL for mometasone furoate. The method was validated according to the ICH guidelines then applied successfully to determine the mentioned drugs in their pharmaceutical preparation and spiked human plasma samples. For plasma samples, the results showed that the method can determine nadifloxacin, mometasone furoate and miconazole nitrate in human plasma samples with high accuracy and precision. Introduction Nadifloxacin (NAD), (RS)-9-fluoro-8-(4-hydroxypiperidin-1-yl)-5-methyl-1-oxo-6,7-dihydro-1H,5H-pyrido[3,2,1-ij]quinoline-2-carboxylic acid (Figure 1a), is a second-generation fluoroquinolone antibiotic with a molecular weight of 360.385 g/mol. It acts by inhibiting the activity of both DNA gyrase and topoisomerase IV, which are essential enzymes for bacterial DNA replication. NAD can be used for treatment of Methicillin-resistant Staphylococcus aureus (MRSA), coagulase-negative staphylococci and anaerobic bacteria [1]. It is approved to be used in acne treatment and skin infections [2, 3]. Mometasone furoate (MF), 9a,21-dichloro-11b, 17-dihydroxy16a-methylpregna-1,4-diene-3,20-dione 17-(2-furoate) (Figure 1b), is a topical corticosteroid with a molecular weight of 521.431 g/mol. It acts by inhibiting the release of phospholipase A2, a principle enzyme for the formation of arachidonic acid–based inflammatory mediators prostaglandins and leukotrienes [4]. Miconazole nitrate (MN) [l-(2,4-dichloro-β-((2,4-chlorobenzyl)oxy)phenethyl)imidazole] (Figure 1c) is a synthetic imidazole derivative with a molecular weight of 479.135 g/mol. It acts by decreasing the ability of fungi to produce ergosterol which is an important part of its cell membrane. It is effective as a topical applicant in treating skin and nail infections and in vaginal candidiasis [5, 6]. The three drugs are formulated together in a pharmaceutical preparation used for treatment of skin infections such as acne vulgaris, athlete’s foot, tinea and folliculitis. Figure 1 Open in new tabDownload slide Chemical structures of nadifloxacin (a), mometasone furoate (b) and miconazole nitrate (c). 338 × 190 mm (300 × 300 DPI). Figure 1 Open in new tabDownload slide Chemical structures of nadifloxacin (a), mometasone furoate (b) and miconazole nitrate (c). 338 × 190 mm (300 × 300 DPI). A literature survey showed different methods for analysis of NAD such as chromatographic methods using high-performance liquid chromatography (HPLC) [7, 8], high-performance thin-layer chromatography (HPTLC) [9] and spectrophotometric methods [10, 11]. As for MN HPLC methods [12, 13], LC/MS method [14], HPTLC method [15] and spectrophotometric method [16] have been reported, while, for MF HPLC methods [17–22], spectrophotometric methods [23] have been published. Regarding the three drugs in combination, only the HPTLC method has been reported in the literature [2]. HPLC is the most commonly preferred method of analysis of mixtures in quality control laboratories. So the aim of this work was to develop a UPLC method for separation and quantification of the ternary mixture in bulk powder, pharmaceutical preparation and spiked human plasma. To our knowledge, it is the first time in literature that the ternary mixture in human plasma was determined. Experimental Materials and reagents NAD, MF and MN standards were supplied from EVA Pharmaceuticals (Cairo, Egypt). All the standards were certified to contain 99.99%. Nadibact Plus® Cream manufactured by Cipla was purchased from the Egyptian market. It contains 1% (w/w) NAD, 0.1% (w/w) MF and 2% (w/w) MN. Human plasma was supplied from Vacsera, Giza, Egypt. HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Loughborough, Leicestershire, UK). Analytical grade ammonium acetate, perchloric acid and glacial acetic acid were purchased from Fisher Scientific (Loughborough, Leicestershire, UK). Instrumentation The UPLC system used was a Thermo Fisher UHPLC Dionex UltiMate 3000 (Germering, Germany). It consists of a pump (ISO-3100SD), autosampler (WPS 3000 SL), column thermostat (TCC-3000SD) and diode array detector (DAD-3000 RS) (Germering, Germany). The software was Chromeleon 6.8 (Germering, Germany). pH was measured using a pH meter (Jenway pH meter 3310, Dunmow, Essex, UK). MilliQ water was obtained from a water purification system (Thermo Scientific Barnstead Smart2Pure 3 UV, Hungary). An ultra sonicator was used for drug extraction from the pharmaceutical preparations (Elmasonic S 60 (H), Germany). Plasma samples were vortexed by (VELP Scientifica, Europe), centrifuged using the Centurion K241R centrifuge (UK) and then evaporated using a rotary vacuum concentrator with a vacuum pump (DVP TYRO 12, Germany), solvent trap (CHRIST CT 02-50, Germany) and rotor (CHRIST RVC 2-18 CDplus, Germany). Chromatographic conditions Separation was performed using an ACQUITY UPLC C18 column of 2.2 μm particle size (2.1 × 100 mm) as a stationary phase, and the mobile phase consists of methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate with pH adjusted to (7.00) using acetic acid; separation was done in the isocratic mode, at a flow rate of 0.6 mL/min, and the column oven was adjusted to 30°C and UV detection at 220 nm. Standard solutions A standard stock solution (1 mg/mL) for each drug was prepared by weighing accurately 25.0 mg of the standard drug powder in a 25-mL volumetric flask. The drugs were dissolved and brought to volume with methanol. Working solutions were prepared by making separate dilutions from each stock solution using methanol, so that the final concentrations were 5, 10, 20, 50, 100, 200, 300, 400 and 500 μg/mL for each drug. Stock solutions were stored at 4°C, when not in use. Method Validation Validation was done according to ICH guidelines [24]. Linearity Linearity of the method was determined by preparing 5, 10, 20, 50, 100, 200, 300, 400 and 500 μg/mL of each drug in methanol. Then, samples were injected in the UPLC. The peak area was determined for each drug and plotted against the corresponding concentration. Selectivity Selectivity is the ability of the method to separate NAD, MF and MN at baseline with sharp uniform peaks, without interference from the excipients of the pharmaceutical preparation or from the plasma matrix. Selectivity was determined by the peak purity test. Limit of detection (LOD) and limit of quantification (LOQ) LOD is the lowest concentration that can be detected compared to the baseline noise while, LOQ is the smallest concentration that can be quantified. LOD and LOQ can be determined by the following equations, where S/N is the signal-to-noise ratio. $$\mathrm{LOD}=3\times \frac{\mathrm{S}}{\mathrm{N}}$$ $$\mathrm{LOQ}=10\times \frac{\mathrm{S}}{\mathrm{N}}$$ Precision Precision is the closeness of the results obtained from analysis to each other. Inter-day precision and intra-day precision were evaluated using three concentrations 10, 50 and 100 μg/mL; in the intra-day precision, each concentration was injected three times in the same day, while for the inter-day precision, each concentration was measured in three different days. Results were interpreted by % relative standard deviation (% RSD). Table I Linearity, Limit of Detection (LOD) and Limit of Quantification (LOQ) of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate . Regression equationa . R2 . LOD (μg/mL) . LOQ (μg/mL) . Nadifloxacin |$Y=0.9932$|X+4.4136 0.9996 1 5 Mometasone furoate |$Y=0.2787$|X-0.339 0.9994 5 20 Miconazole nitrate |$Y=0.4737X+1.4869$| 0.9999 1 5 . Regression equationa . R2 . LOD (μg/mL) . LOQ (μg/mL) . Nadifloxacin |$Y=0.9932$|X+4.4136 0.9996 1 5 Mometasone furoate |$Y=0.2787$|X-0.339 0.9994 5 20 Miconazole nitrate |$Y=0.4737X+1.4869$| 0.9999 1 5 aAverage of three times Open in new tab Table I Linearity, Limit of Detection (LOD) and Limit of Quantification (LOQ) of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate . Regression equationa . R2 . LOD (μg/mL) . LOQ (μg/mL) . Nadifloxacin |$Y=0.9932$|X+4.4136 0.9996 1 5 Mometasone furoate |$Y=0.2787$|X-0.339 0.9994 5 20 Miconazole nitrate |$Y=0.4737X+1.4869$| 0.9999 1 5 . Regression equationa . R2 . LOD (μg/mL) . LOQ (μg/mL) . Nadifloxacin |$Y=0.9932$|X+4.4136 0.9996 1 5 Mometasone furoate |$Y=0.2787$|X-0.339 0.9994 5 20 Miconazole nitrate |$Y=0.4737X+1.4869$| 0.9999 1 5 aAverage of three times Open in new tab Figure 2 Open in new tabDownload slide UPLC chromatogram of 200, 20 and 400 μg/mL of NAD, MF and MN standards (a), respectively, pharmaceutical preparation (b) and spiked plasma sample (c). Eluted under the optimum conditions: ACQUITY UPLC C18 column of 2.2 μm particle size (2.1 × 100 mm) as a stationary phase, methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate, then the pH was adjusted to (7.00) using acetic acid as a mobile phase, at a flow rate of 0.6 mL/min; the column oven was adjusted at 30°C; and the UV detection was at 220 nm. Figure 2 Open in new tabDownload slide UPLC chromatogram of 200, 20 and 400 μg/mL of NAD, MF and MN standards (a), respectively, pharmaceutical preparation (b) and spiked plasma sample (c). Eluted under the optimum conditions: ACQUITY UPLC C18 column of 2.2 μm particle size (2.1 × 100 mm) as a stationary phase, methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate, then the pH was adjusted to (7.00) using acetic acid as a mobile phase, at a flow rate of 0.6 mL/min; the column oven was adjusted at 30°C; and the UV detection was at 220 nm. Accuracy Accuracy was evaluated by spiking the dosage form with three different concentrations of each drug, then the recovery percentage of the added concentrations of the standard drugs was evaluated. Robustness Robustness is the ability of the method to remain unaffected due to small changes in chromatographic conditions. In this method, the conditions studied were temperature, % aqueous: organic phase (% Aq: Org) of the mobile phase, pH of the mobile phase, wavelength and flow rate changes. Results were interpreted by % RSD. Analysis of pharmaceutical preparation One gram of Nadibact Plus® Cream (equivalent to 10 mg NAD, 1 mg MF and 20 mg MN) was weighed and sonicated with 8 mL methanol for 20 min; the volume was completed to 10 mL with methanol to obtain a stock solution with concentrations of 1, 0.1 and 2 mg/mL NAD, MF and MN, respectively. The sample was filtered using a 0.45-μm Whatman paper. The stock solution was then diluted with methanol to prepare the required concentration. Analysis of spiked human plasma samples Working solutions with concentration of 300 μg/mL were prepared from NAD, MF and MN standard stock solutions. One milliliter from each working solution was mixed together, so that the final concentration was 100 μg/mL for each drug. Then, 200, 300, 400, 500 and 1000 μL were taken from this mixture and added separately to 0.5 mL plasma to prepare 40, 60, 80, 100 and 200 μg/mL plasma for each drug, and 1.5 mL acetonitrile was added. The mixture was vortexed for 3 min at 3000 RPM, centrifuged at 6000 RPM for 15 min. The supernatant was collected and evaporated to dryness using a rotatory vacuum concentrator at 1500 RPM and 60°C for 2 h. Samples were reconstituted using 1 mL methanol then injected to the UPLC. Table II Intra-Day and Inter-Day Precision of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate Drug . Concentration (μg/mL) . Intra-day precisiona % RSD . Inter-day precisiona % RSD . Nadifloxacin 10 1.55 0.77 50 1.12 1.33 100 0.26 0.47 Mometasone furoate 10 0.26 0.29 50 0.17 0.39 100 0.07 0.14 Miconazole nitrate 10 0.85 0.45 50 0.02 0.09 100 0.02 0.05 Drug . Concentration (μg/mL) . Intra-day precisiona % RSD . Inter-day precisiona % RSD . Nadifloxacin 10 1.55 0.77 50 1.12 1.33 100 0.26 0.47 Mometasone furoate 10 0.26 0.29 50 0.17 0.39 100 0.07 0.14 Miconazole nitrate 10 0.85 0.45 50 0.02 0.09 100 0.02 0.05 aAverage of three times Open in new tab Table II Intra-Day and Inter-Day Precision of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate Drug . Concentration (μg/mL) . Intra-day precisiona % RSD . Inter-day precisiona % RSD . Nadifloxacin 10 1.55 0.77 50 1.12 1.33 100 0.26 0.47 Mometasone furoate 10 0.26 0.29 50 0.17 0.39 100 0.07 0.14 Miconazole nitrate 10 0.85 0.45 50 0.02 0.09 100 0.02 0.05 Drug . Concentration (μg/mL) . Intra-day precisiona % RSD . Inter-day precisiona % RSD . Nadifloxacin 10 1.55 0.77 50 1.12 1.33 100 0.26 0.47 Mometasone furoate 10 0.26 0.29 50 0.17 0.39 100 0.07 0.14 Miconazole nitrate 10 0.85 0.45 50 0.02 0.09 100 0.02 0.05 aAverage of three times Open in new tab Table III Accuracy: Dosage Form Spiked with Different Concentrations of Each Drug Standard Drug . Prepared concentration (μg/mL) . Found concentration (μg/mL)a . R% a . RSD . Nadifloxacin 110 109.79 99.81 0.81 120 120.09 100.08 0.098 130 130.30 100.23 0.05 Mometasone furoate 20 19.99 99.99 0.04 30 30.13 100.43 0.34 40 40.38 100.95 0.68 Miconazole Nitrate 210 211.76 100.84 0.17 220 220.73 100.33 0.01 230 228.80 99.48 0.77 Drug . Prepared concentration (μg/mL) . Found concentration (μg/mL)a . R% a . RSD . Nadifloxacin 110 109.79 99.81 0.81 120 120.09 100.08 0.098 130 130.30 100.23 0.05 Mometasone furoate 20 19.99 99.99 0.04 30 30.13 100.43 0.34 40 40.38 100.95 0.68 Miconazole Nitrate 210 211.76 100.84 0.17 220 220.73 100.33 0.01 230 228.80 99.48 0.77 aAverage of three times Open in new tab Table III Accuracy: Dosage Form Spiked with Different Concentrations of Each Drug Standard Drug . Prepared concentration (μg/mL) . Found concentration (μg/mL)a . R% a . RSD . Nadifloxacin 110 109.79 99.81 0.81 120 120.09 100.08 0.098 130 130.30 100.23 0.05 Mometasone furoate 20 19.99 99.99 0.04 30 30.13 100.43 0.34 40 40.38 100.95 0.68 Miconazole Nitrate 210 211.76 100.84 0.17 220 220.73 100.33 0.01 230 228.80 99.48 0.77 Drug . Prepared concentration (μg/mL) . Found concentration (μg/mL)a . R% a . RSD . Nadifloxacin 110 109.79 99.81 0.81 120 120.09 100.08 0.098 130 130.30 100.23 0.05 Mometasone furoate 20 19.99 99.99 0.04 30 30.13 100.43 0.34 40 40.38 100.95 0.68 Miconazole Nitrate 210 211.76 100.84 0.17 220 220.73 100.33 0.01 230 228.80 99.48 0.77 aAverage of three times Open in new tab Results Chromatographic conditions Different chromatographic conditions were investigated. First, the mobile phase acetonitrile: water (80:20; v/v) was tried but the NAD peak was not sharp and uniform while MN and MF peaks were tailed. The increasing acetonitrile ratio did not enhance the peak shape. Also, increasing the aqueous phase volume was not a good choice as NAD is poorly soluble in water. Triethylamine (0.1%) was added to improve the separation and peak shape, and triethylamine blocks underivatized silanol groups which would strongly interact with molecules of interest, but the peaks were tailed and not uniform. Table IV Robustness of the Method Used for Nadifloxacin, Mometasone Furoate and Miconazole Nitrate Analysis to Different Investigated Factors Effect of temperature change . Temperature change (°C) % RSDa Nadifloxacin 30 ± 1 0.262–0.55 Mometasone furoate 0.74–0.90 Miconazole nitrate 0.28–0.65 Effect of % aqueous: organic phase change % Aqueous: organic % RSDa Nadifloxacin 30 ± 1:70 ± 1 0.14–0.54 Mometasone furoate 0.28–0.72 Miconazole nitrate 0.21–0.55 Effect of pH change pH % RSDa Nadifloxacin 7 ± 0.1 0.22–0.58 Mometasone furoate 0.66–1.06 Miconazole nitrate 0.26–0.55 Effect of wavelength change Wavelength (nm) % RSDa Nadifloxacin 220 ± 2 0.23–0.39 Mometasone furoate 0.74–0.96 Miconazole nitrate 0.30–1.56 Effect of flow rate change Flow rate (mL/min) % RSDa Nadifloxacin 1 ± 0.1 0.17–0.35 Mometasone furoate 0.70–1.38 Miconazole nitrate 0.24–1.05 Effect of temperature change . Temperature change (°C) % RSDa Nadifloxacin 30 ± 1 0.262–0.55 Mometasone furoate 0.74–0.90 Miconazole nitrate 0.28–0.65 Effect of % aqueous: organic phase change % Aqueous: organic % RSDa Nadifloxacin 30 ± 1:70 ± 1 0.14–0.54 Mometasone furoate 0.28–0.72 Miconazole nitrate 0.21–0.55 Effect of pH change pH % RSDa Nadifloxacin 7 ± 0.1 0.22–0.58 Mometasone furoate 0.66–1.06 Miconazole nitrate 0.26–0.55 Effect of wavelength change Wavelength (nm) % RSDa Nadifloxacin 220 ± 2 0.23–0.39 Mometasone furoate 0.74–0.96 Miconazole nitrate 0.30–1.56 Effect of flow rate change Flow rate (mL/min) % RSDa Nadifloxacin 1 ± 0.1 0.17–0.35 Mometasone furoate 0.70–1.38 Miconazole nitrate 0.24–1.05 aAverage of three times Open in new tab Table IV Robustness of the Method Used for Nadifloxacin, Mometasone Furoate and Miconazole Nitrate Analysis to Different Investigated Factors Effect of temperature change . Temperature change (°C) % RSDa Nadifloxacin 30 ± 1 0.262–0.55 Mometasone furoate 0.74–0.90 Miconazole nitrate 0.28–0.65 Effect of % aqueous: organic phase change % Aqueous: organic % RSDa Nadifloxacin 30 ± 1:70 ± 1 0.14–0.54 Mometasone furoate 0.28–0.72 Miconazole nitrate 0.21–0.55 Effect of pH change pH % RSDa Nadifloxacin 7 ± 0.1 0.22–0.58 Mometasone furoate 0.66–1.06 Miconazole nitrate 0.26–0.55 Effect of wavelength change Wavelength (nm) % RSDa Nadifloxacin 220 ± 2 0.23–0.39 Mometasone furoate 0.74–0.96 Miconazole nitrate 0.30–1.56 Effect of flow rate change Flow rate (mL/min) % RSDa Nadifloxacin 1 ± 0.1 0.17–0.35 Mometasone furoate 0.70–1.38 Miconazole nitrate 0.24–1.05 Effect of temperature change . Temperature change (°C) % RSDa Nadifloxacin 30 ± 1 0.262–0.55 Mometasone furoate 0.74–0.90 Miconazole nitrate 0.28–0.65 Effect of % aqueous: organic phase change % Aqueous: organic % RSDa Nadifloxacin 30 ± 1:70 ± 1 0.14–0.54 Mometasone furoate 0.28–0.72 Miconazole nitrate 0.21–0.55 Effect of pH change pH % RSDa Nadifloxacin 7 ± 0.1 0.22–0.58 Mometasone furoate 0.66–1.06 Miconazole nitrate 0.26–0.55 Effect of wavelength change Wavelength (nm) % RSDa Nadifloxacin 220 ± 2 0.23–0.39 Mometasone furoate 0.74–0.96 Miconazole nitrate 0.30–1.56 Effect of flow rate change Flow rate (mL/min) % RSDa Nadifloxacin 1 ± 0.1 0.17–0.35 Mometasone furoate 0.70–1.38 Miconazole nitrate 0.24–1.05 aAverage of three times Open in new tab Table V The Penalty Points to Calculate the Analytical Eco-Scale Type of reagent . Penalty points . Methanol (Less than 10 mL) = 18 Acetonitrile (Less than 10 mL) = 8 Acetic acid (Less than 10 mL) = 2 Hazardousness (None) = 0 Energy consumption (Less than or equal to 0.1 kWh per sample) = 0 Waste production (1–10 mL) = 3 Total penalty points 31 Analytical Eco-Scale total score 69 Type of reagent . Penalty points . Methanol (Less than 10 mL) = 18 Acetonitrile (Less than 10 mL) = 8 Acetic acid (Less than 10 mL) = 2 Hazardousness (None) = 0 Energy consumption (Less than or equal to 0.1 kWh per sample) = 0 Waste production (1–10 mL) = 3 Total penalty points 31 Analytical Eco-Scale total score 69 Open in new tab Table V The Penalty Points to Calculate the Analytical Eco-Scale Type of reagent . Penalty points . Methanol (Less than 10 mL) = 18 Acetonitrile (Less than 10 mL) = 8 Acetic acid (Less than 10 mL) = 2 Hazardousness (None) = 0 Energy consumption (Less than or equal to 0.1 kWh per sample) = 0 Waste production (1–10 mL) = 3 Total penalty points 31 Analytical Eco-Scale total score 69 Type of reagent . Penalty points . Methanol (Less than 10 mL) = 18 Acetonitrile (Less than 10 mL) = 8 Acetic acid (Less than 10 mL) = 2 Hazardousness (None) = 0 Energy consumption (Less than or equal to 0.1 kWh per sample) = 0 Waste production (1–10 mL) = 3 Total penalty points 31 Analytical Eco-Scale total score 69 Open in new tab Another mobile phase was investigated; it consists of acetonitrile: tetrahydrofuran: 25 mM phosphate buffer with pH (3.00) (65:5:30; v/v); the aim of using tetrahydrofuran in this mobile phase was to improve the drug solubility and elution from the stationary phase. Phosphate buffer was added to improve the ionization of the drugs and to enhance the separation, but NAD and MF peaks overlapped. The optimum mobile phase was found to be methanol, acetonitrile and water with ratio 50:20:30 (v/v/v) and 0.1 g ammonium acetate with pH adjusted to 7.00 using acetic acid, at a flow rate of 0.6 mL/min. A lower flow rate increases the analysis time, while a higher one leads to an overlap between peaks. Different temperatures were tried, and 30°C was the optimum. UV detection is at 220 nm; the choice of this wavelength depends on λmax obtained from the absorption spectrum using a photodiode array (PDA) detector. Under these conditions, NAD, MF and MN standards eluted at 0.62, 1.3 and 4.85 min, respectively. The total run time is 6 min (Figure 2a). Method Validation Linearity, LOD and LOQ The method is linear in the range 5 to 400 μg/mL for NAD and MN and 20 to 500 μg/mL for MF. LOD and LOQ are presented in Table I. Selectivity The method is selective due to the ability of separation of NAD, MF and MN at baseline without interference from the excipients of dosage form or from the endogenous substances in plasma samples (Figure 2b and c). Precision The results show that the method is precise as the % RSD is less than 2% (Table II). Accuracy The method is accurate as the percentage recovery ranges from 99.48 to 100.95% for the three drugs (Table III). Robustness The factors investigated showed that the method is robust. The method is not adversely affected by the changes previously mentioned as evident from the low values of % RSD (Table IV). Table VI Analysis of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate in Their Pharmaceutical Preparation Drug . Prepared concentration (μg/mL) . Found concentration (μg/mL)a . R %a . % RSD (inter-day) . % RSD (intra-day) . Nadifloxacin 200 201.2 100.6 0.29 0.45 Mometasone furoate 20 20.05 102.5 0.26 0.38 Miconazole nitrate 400 402 100.5 0.28 0.28 Drug . Prepared concentration (μg/mL) . Found concentration (μg/mL)a . R %a . % RSD (inter-day) . % RSD (intra-day) . Nadifloxacin 200 201.2 100.6 0.29 0.45 Mometasone furoate 20 20.05 102.5 0.26 0.38 Miconazole nitrate 400 402 100.5 0.28 0.28 aAverage of three times Open in new tab Table VI Analysis of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate in Their Pharmaceutical Preparation Drug . Prepared concentration (μg/mL) . Found concentration (μg/mL)a . R %a . % RSD (inter-day) . % RSD (intra-day) . Nadifloxacin 200 201.2 100.6 0.29 0.45 Mometasone furoate 20 20.05 102.5 0.26 0.38 Miconazole nitrate 400 402 100.5 0.28 0.28 Drug . Prepared concentration (μg/mL) . Found concentration (μg/mL)a . R %a . % RSD (inter-day) . % RSD (intra-day) . Nadifloxacin 200 201.2 100.6 0.29 0.45 Mometasone furoate 20 20.05 102.5 0.26 0.38 Miconazole nitrate 400 402 100.5 0.28 0.28 aAverage of three times Open in new tab Table VII Analysis of Plasma Samples Spiked with Different Concentrations of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate Drug . Prepared concentration (μg/mL plasma) . Found concentration (μg/mL plasma) a . R %a . % RSD Intra-day . % RSD Inter-day . Nadifloxacin 40 40.4 101 0.21 0.41 60 59.78 99.63 0.24 0.60 80 80.1 100.13 0.25 0.35 100 100.04 100.04 0.11 0.60 200 200.2 100.1 0.12 0.10 Mometasone furoate 40 39.96 99.9 1.53 1.12 60 60.08 100.13 0.56 1.87 80 80.6 100.8 0.40 0.92 100 100.6 100.75 0.39 1.13 200 199.94 99.97 0.18 0.36 Miconazole nitrate 40 40.24 100.6 0.41 0.97 60 60.4 100.67 0.17 0.63 80 79.16 98.95 0.18 0.52 100 100.06 100.06 0.20 0.40 200 200.4 100.2 0.05 0.33 Drug . Prepared concentration (μg/mL plasma) . Found concentration (μg/mL plasma) a . R %a . % RSD Intra-day . % RSD Inter-day . Nadifloxacin 40 40.4 101 0.21 0.41 60 59.78 99.63 0.24 0.60 80 80.1 100.13 0.25 0.35 100 100.04 100.04 0.11 0.60 200 200.2 100.1 0.12 0.10 Mometasone furoate 40 39.96 99.9 1.53 1.12 60 60.08 100.13 0.56 1.87 80 80.6 100.8 0.40 0.92 100 100.6 100.75 0.39 1.13 200 199.94 99.97 0.18 0.36 Miconazole nitrate 40 40.24 100.6 0.41 0.97 60 60.4 100.67 0.17 0.63 80 79.16 98.95 0.18 0.52 100 100.06 100.06 0.20 0.40 200 200.4 100.2 0.05 0.33 aAverage of three times Open in new tab Table VII Analysis of Plasma Samples Spiked with Different Concentrations of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate Drug . Prepared concentration (μg/mL plasma) . Found concentration (μg/mL plasma) a . R %a . % RSD Intra-day . % RSD Inter-day . Nadifloxacin 40 40.4 101 0.21 0.41 60 59.78 99.63 0.24 0.60 80 80.1 100.13 0.25 0.35 100 100.04 100.04 0.11 0.60 200 200.2 100.1 0.12 0.10 Mometasone furoate 40 39.96 99.9 1.53 1.12 60 60.08 100.13 0.56 1.87 80 80.6 100.8 0.40 0.92 100 100.6 100.75 0.39 1.13 200 199.94 99.97 0.18 0.36 Miconazole nitrate 40 40.24 100.6 0.41 0.97 60 60.4 100.67 0.17 0.63 80 79.16 98.95 0.18 0.52 100 100.06 100.06 0.20 0.40 200 200.4 100.2 0.05 0.33 Drug . Prepared concentration (μg/mL plasma) . Found concentration (μg/mL plasma) a . R %a . % RSD Intra-day . % RSD Inter-day . Nadifloxacin 40 40.4 101 0.21 0.41 60 59.78 99.63 0.24 0.60 80 80.1 100.13 0.25 0.35 100 100.04 100.04 0.11 0.60 200 200.2 100.1 0.12 0.10 Mometasone furoate 40 39.96 99.9 1.53 1.12 60 60.08 100.13 0.56 1.87 80 80.6 100.8 0.40 0.92 100 100.6 100.75 0.39 1.13 200 199.94 99.97 0.18 0.36 Miconazole nitrate 40 40.24 100.6 0.41 0.97 60 60.4 100.67 0.17 0.63 80 79.16 98.95 0.18 0.52 100 100.06 100.06 0.20 0.40 200 200.4 100.2 0.05 0.33 aAverage of three times Open in new tab Figure 3 Open in new tabDownload slide UPLC chromatogram represents a blank plasma sample under the optimum conditions: ACQUITY UPLC C18 column of 2.2 μm particle size (2.1 × 100 mm) as a stationary phase, methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate, then the pH was adjusted to (7.00) using acetic acid as a mobile phase, at a flow rate of 0.6 mL/min; the column oven was adjusted at 30°C; and the UV detection was at 220 nm. Figure 3 Open in new tabDownload slide UPLC chromatogram represents a blank plasma sample under the optimum conditions: ACQUITY UPLC C18 column of 2.2 μm particle size (2.1 × 100 mm) as a stationary phase, methanol, acetonitrile and water with ratio (50:20:30; v/v/v) and 0.1 g ammonium acetate, then the pH was adjusted to (7.00) using acetic acid as a mobile phase, at a flow rate of 0.6 mL/min; the column oven was adjusted at 30°C; and the UV detection was at 220 nm. Assessment of Greenness of the Method: The Analytical Eco-Scale The analytical Eco-Scale is one good, semi-quantitative tool used for the assessment of greenness of the analytical methods. It compares the various parameters and steps for the whole analytical process. The analytical Eco-Scale is calculated by using penalty points to any factor in the analytical procedure that disagrees or does not match with perfect green analysis [25]. The result of calculation is ranked on a scale, where more than 75 represents excellent green analysis, more than 50 represents acceptable green analysis and less than 50 represents inadequate green analysis [26]. Penalty points are calculated based on four main parameters of the analytical procedure: amount of reagents, hazardousness, energy consumption and waste production. As presented in Table V, the total Eco-scale was 69. So, the results showed that the method represents acceptable green analysis. Application of the method to the pharmaceutical preparation and spiked human plasma samples Sample preparation is important in completely extracting the drugs and reducing the effect of excipients in pharmaceutical preparation as well as in reducing the effect of plasma proteins and other components from the plasma matrix that can potentially interfere with the analyte of interest and damage the analytical column. Analysis of the pharmaceutical preparation Different solvents such as water, methanol and acetonitrile were investigated to extract the three drugs, under investigation, from the pharmaceutical preparation. The solvent that shows the maximum %R is methanol. Accordingly, it is selected to be the extraction solvent. The suggested method was applied to determine the three drugs in their dosage form (Table VI). Analysis of spiked human plasma samples For protein precipitation, first perchloric acid was examined by adding 2 mL of the acid to different volumes of plasma, but all the precipitates dissolved due to the high strength of the acid. As the polarity of an organic solvent increases, it becomes a less effective precipitating agent [27, 28]. Methanol is more polar than acetonitrile and is therefore expected to be less effective in precipitating plasma proteins; in the current study, acetonitrile was tried to precipitate plasma proteins, where best results were obtained upon using 0.5 mL of plasma and 1.5 mL of acetonitrile. A blank plasma chromatogram is evidence that using acetonitrile and plasma sample with this ratio was the best in the removal any interfering substance, as the noise is less 1% of the drug peak (Figure 3). A combination of larger volumes of acetonitrile and 0.5 mL of plasma was also tried, but this results in the formation of a turbid supernatant following the centrifugation step. The use of equal volumes of acetonitrile and plasma can lead to precipitation of about 95% of the proteins [29]. The method is precise and accurate for the determination of the three drugs in plasma (Table VII). Discussion The proposed method was developed and validated for the analysis of NAD, MF and MN. It can be used for the analysis of the ternary mixture in their combined dosage form. Also, it can be used for the analysis of the ternary mixture in human plasma samples. To the best of our knowledge, this is the first UPLC method for the analysis of the three drugs in human plasma. The method was successfully validated according to ICH guidelines. The method showed high selectivity; it can separate the three drugs without interference from the plasma proteins and dosage form excipients. The method showed high accuracy and precision. This method was developed as a preliminary study to a pharmacokinetic study. Conclusion The suggested method is considered the first RP-UPLC validated method for the analysis of NAD, MF and MN; this method offers several advantages such as good resolution in a small run time (6 min), an isocratic mobile phase which can be used, and the method represents acceptable green analysis. 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TI - A Validated Ultra-Performance Liquid Chromatographic Method for the Simultaneous Determination of Nadifloxacin, Mometasone Furoate and Miconazole Nitrate in Their Combined Dosage Form and Spiked Human Plasma Samples
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmz082
DA - 2020-01-17
UR - https://www.deepdyve.com/lp/oxford-university-press/a-validated-ultra-performance-liquid-chromatographic-method-for-the-AWsPmXKVuU
SP - 867
VL - 57
IS - 10
DP - DeepDyve
ER -