Simultaneous ecofriendly HPLC determination of clotrimazole, miconazole, metronidazole, and lidocaine in pharmaceutical dosage forms

Abstract

The analysis of multicomponent pharmaceutical formulations containing drugs with significant diverse physicochemical properties, like 6 orders of magnitude of pKa values, which permanently represents a significant challenge in modern pharmaceutical analysis. The simultaneous determination of multiple active agents offers substantial advantages by reducing analysis time and resource consumption. Furthermore, the increased analytical demand during health emergencies highlights the necessity for validated, rapid, and reliable methods capable of supporting both therapeutic assessment and regulatory decision-making. In the present study, a reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and optimized for the simultaneous determination of four analytes: clotrimazole, miconazole, metronidazole, and lidocaine. Various approaches were investigated for mobile phase composition and column selection to achieve optimal separation and accurate quantitative determination. Chromatographic separation was examined using several columns, including Waters Spherisorb CN (250 × 4.6 mm, 5 µm), LiChrospher 100 CN (125 × 4 mm, 5 µm), LiChrospher 100 CN (250 × 4 mm, 5 µm), Zorbax SB CN (150 × 4.6 mm, 3.5 µm and 250 × 4.6 mm, 5 µm), Zorbax Rx C8 (250 × 4.6 mm, 5 µm), and Zorbax SB Phenyl (150 × 4.6 mm, 3.5 µm). Isocratic elution was employed using mixtures of acetonitrile, methanol, and phosphoric acid or trifluoroacetic acid in different proportions at flow rates of 1.0 and 1.2 mL min^− 1. UV-detection was carried out at 215 nm. The developed method demonstrated excellent system suitability and complied with ICH Q2 (R2) validation requirements, showing satisfactory linearity (R² > 0.999), accuracy (recoveries within 98–102%), and precision (RSD < 2%). In addition, the sustainability of the proposed method was comprehensively assessed using AGREE, MoGAPI, and BAGI tools to evaluate its greenness, whiteness, blueness and applicability. Overall, the method provides a robust, efficient, and sustainable analytical platform for quality control laboratories, enabling reliable routine analysis of multicomponent pharmaceutical products and therapeutic surveillance.

Introduction

Infectious diseases constitute a major contributor to global mortality. Given the broad spectrum of pathogens affecting male populations, particular emphasis has been placed on protozoal, fungal, and bacterial infections [1]. Within this context, three agents with distinct biological activities, namely clotrimazole, miconazole, and metronidazole and one local anesthetic, lidocaine, were subjected to comprehensive chemical characterization. Clotrimazole is a synthetic antifungal agent belonging to the class of imidazole derivatives. Chemically, clotrimazole (1-[(2-chlorophenyl)diphenylmethyl]-1H-imidazole) is practically insoluble in water with a log P value of 6.1 and a pKa of 4.1 [2, 3]. The European Pharmacopoeia (Eur. Ph.) monograph for clotrimazole specifies non aqueous acidimetry for assay determination and liquid chromatography for related substances using end-capped, extra-densely bonded octylsilyl silica gel (5 µm). Gradient elution is employed with a total run time of 30 min [2]. Miconazole is also a synthetic antifungal agent of the imidazole class. Miconazole (1-[(2RS)-2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy]ethyl]-1H-imidazole) is very slightly soluble in water, freely soluble in methanol, and soluble in ethanol, with a log P value of 5.96 and a pKa of 6.48 [2, 4]. The Eur. Ph. includes monographs for both miconazole and miconazole nitrate, recommending nonaqueous acidimetry for assay determination and liquid chromatography for related substances using end-capped octylsilyl silica gel (3 µm) [2]. Metronidazole is a widely used antimicrobial agent belonging to the nitroimidazole class of chemotherapeutics, with potent activity against obligate anaerobic microorganisms. Clinically, it is commonly employed in the treatment of infections caused by Bacteroides, Clostridium, and Fusobacterium species, as well as Peptococcus and Peptostreptococcus species. Metronidazole (2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanol) is slightly soluble in water, acetone, ethanol (96%), and methylene chloride, with a log P value of − 0.02 and pKa values of 2.57 and 15.42 [2, 5]. The Eur. Ph. monograph for metronidazole describes nonaqueous acidimetry for assay determination and liquid chromatography for related substances using octadecylsilyl silica gel (5 µm). The mobile phase consists of methanol and an aqueous solution of potassium dihydrogen phosphate, with detection at 315 nm and a flow rate of 1.0 mL min^− 1 [2]. Lidocaine (2-(diethylamino)- N-(2,6-dimethylphenyl)acetamide) is a local anesthetic that is practically insoluble in water but very soluble in ethanol and methylene chloride; its hydrochloride salt is freely soluble in water. Lidocaine has a log P value of 2.84 and pKa values of 7.75 and 13.78 [2, 6]. The Eur. Ph. monograph for lidocaine recommends non aqueous acidimetry for assay determination and liquid chromatography for related substances using end-capped, polar-embedded octadecylsilyl amorphous organosilica polymer (5 µm) [2].

Several high-performance liquid chromatography (HPLC) methods have been developed for the determination of these drugs individually or in partial combinations; however, no method has been reported for the simultaneous determination of all four analytes [7–27]. Consequently, alternative HPLC approaches for the concurrent analysis of clotrimazole, miconazole, metronidazole, and lidocaine are warranted to improve analytical efficiency, accuracy, and resource utilization while reducing analysis time. The objective of the present study was to develop and validate a robust and accurate HPLC method for the simultaneous quantification of clotrimazole, miconazole, metronidazole, and lidocaine in pharmaceutical dosage forms. This approach enables the separation and determination of all four compounds within a single chromatographic run, significantly decreasing solvent consumption and analysis time compared with conventional individual methods. Furthermore, the sustainability of the proposed method was evaluated using established greenness, whiteness, blueness assessment tools, including AGREE, MoGAPI, BAGI, confirming its compliance with modern principles of environmentally responsible and resource-efficient analytical science.

Experimental

Instrumentation

The following HPLC columns were used: Luna C8 (150 × 4.6 mm 3 μm), Luna C18 (100 × 4.6 mm 3 μm), InertClone ODS (150 × 4.6 mm 3.5 μm) purchased from Phenomenex; Zorbax SB CN (150 × 4.6 mm 3.5 μm) and (250 × 4.6 mm 5 μm), Zorbax Rx C8 (250 × 4.6 mm 5 μm) and Zorbax SB Phenyl (150 × 4.6 mm 3.5 μm) purchased from Agilent Technologies; LiChrospher 100 CN (125 × 4 mm 5 μm), LiChrospher 100 CN (250 × 4 mm 5 μm) purchased from MERCK; Waters Spherisorb CN 250 × 4.6 mm 5 µm purchased from Waters.

In this research, the instrument used was Shimadzu LC-2050C with diode array detector (DAD) using LabSolutions software (Japan).

Additional equipments used include: analytical RADWAG AS 200/C (Poland), pH meter (Mettler-Toledo, model LE438, Switzerland) and ultrasonic bath (Elmasonic Easy 40 H, Germany). The regenerated nylon (NY) 0.2 µm and 0.45 µm, regenerated cellulose (RC) 0.45 µm, polytetrafluoroethylene (PTFE) 0.2 µm and 0.45 µm syringe filters were purchased from Phenomenex.

Reagents and standards

Dosage forms of clotrimazole (solution 1% Borshchahivskyy khimiko-farmatsevtychnyy zavod,, Ukraine, excipients—propylene glycol, polyethylene glycol 400 (PEG 400), ethanol) miconazole (miconazole 20 mg/g cream, Darnitsa, Ukraine, excipients—propylene glycol, macrogol 400, poloxamer, cetostearyl macrogol ether, cetostearyl alcohol), metronidazole (metronidazole 0.1 g, Monpharm, Ukraine, excipient—hard fat, Limenda – 750 mg of metronidazole and 200 mg of miconazole nitrate, World Medicine İlaç San. ve Tic. A.Ş., Turkey, excipient: Witepsol S55), lidocaine (Limenda L – 750 mg of metronidazole, 200 mg of miconazole nitrate, and 100 mg of lidocaine, World Medicine İlaç San. ve Tic. A.Ş., Turkey, excipients—cetostearyl polyethylene glycol ether, Witepsol S55) were purchased from the local market. Reference standards of the active pharmaceutical ingredient of clotrimazole, miconazole, metronidazole, lidocaine (purity ≥ 99.0% by HPLC) were obtained from USP and Sigma-Aldrich.

The chemicals used—methanol (MeOH), acetonitrile (ACN), phosphoric acid (H₃PO₄), trifluoroacetic acid (TFA), formic acid (HCOOH)—were of gradient grade and purchased from Merck (Darmstadt, Germany). Demineralized water was purchased from Honeywell, Riedel–de Haen (Germany) and had a conductivity of less than 0.5 µS/cm.

Standard solutions

Preparation of stock standard solutions

Stock standard solutions of clotrimazole (1 mg/mL), miconazole (2 mg/mL), metronidazole (7.5 mg/mL), lidocaine (1.0 mg/mL) were prepared separately dissolving of each drug into 100 mL volumetric flasks, each containing 75 mL of MeOH. The contents of each flask were sonicated to ensure complete dissolution. Then the volume was completed with MeOH. A volume of 1 mL was transferred from each flask into a 10 mL volumetric one to prepare a standard solution mixture of the four analytes.

Preparation of working standard solutions

To prepare a working standard solution (clotrimazole (0.1 mg/mL), miconazole (0.2 mg/mL), metronidazole (0.75 mg/mL), lidocaine (0.1 mg/mL)) 1 mL of each stock solution (clotrimazole (1 mg/mL), miconazole (2 mg/mL), metronidazole (7.5 mg/mL), lidocaine (1.0 mg/mL)) was diluted with MeOH into a 10 mL flask.

Chromatographic conditions

Isocratic chromatographic separation was performed on C18, CN, Phenyl columns maintained at 40–42 ± 2 °C. The mobile phase consisted of ACN, MeOH and 0.05% Acid (H₃PO₄, TFA, HCOOH) in the ratio of 35:30:35% v/v. The mobile phase was degassed ultrasonically prior to use. The flow rate 1.0 mL/min, the injection volume was 1–5 μL. UV detection was performed at 215 nm with a total run time of only 6 min.

Procedures

Working standard solutions (clotrimazole (0.1 mg/mL), miconazole (0.2 mg/mL), metronidazole (0.75 mg/mL), lidocaine (0.1 mg/mL)) were transferred into a series of 10 mL volumetric flasks. All flasks were made up to the mark with MeOH to produce final concentrations for each analyte. The studies were performed under optimized chromatographic conditions 2.4. Calibration curves were constructed in the coordinates of the dependence of peak areas and the analyte concentration in μg/mL. Regression equations were calculated for each analyte.

Application to drug analysis

Assay of cream

A precise weight of the cream corresponding to 200 mg miconazole was moved into 100 mL calibrated flask, then 70 mL MeOH was inserted and the mixture was sonicated till thorough dispersal of the cream. The solution was then cooled, brought about to volume with MeOH filtered then injected to the column for analysis.

Assay of suppositories

10 suppositories were accurately weighed and then melted in a water bath at 40 °C until a uniform mass was obtained. The melt was allowed to solidify under continuous stirring to ensure homogeneity. An accurately weighed portion equivalent to one suppository was then transferred into a 100 mL volumetric flask using about 70 mL of MeOH. The mixture was sonicated at 40 °C for 10 min, cooled, and diluted to volume with MeOH. Subsequently, 1 mL of the resulting solution was transferred into a 10 mL volumetric flask and diluted to the mark with MeOH. Appropriate aliquots of the working solutions were filtered through a 0.45 µm membrane filter prior to injection for chromatographic analysis. The concentrations were calculated using simultaneously prepared external standards.

For the standard addition approach, aliquots of each working solution were spiked with known amounts of the standard solutions of the studied compounds to obtain final concentrations within the specified ranges. These samples were then analyzed following the same procedure. The concentrations were determined by comparing the analytical response before and after the addition of the standards [28].

Laboratory prepared mixtures

To prepare different combinations with varying proportions of clotrimazole, metronidazole, miconazole and lidocaine, precise portions of the working solutions were pipetted into multiple 10 mL volumetric flasks, brought to volume with MeOH, and thoroughly homogenized.

Results and discussion

Method development

The selection of chromatographic conditions for the quantitative determination of clotrimazole, metronidazole, miconazole, or lidocaine in single preparations is not a difficult task; however, it becomes more complicated when determining them simultaneously for routine quality control purposes while ensuring excellent chromatographic system suitability parameters, with sufficient accuracy, reproducibility, and eco-friendliness. Clotrimazole and miconazole are imidazole derivatives, lipophilic agents, whereas metronidazole is a nitroimidazole derivative and more hydrophilic. Lidocaine has a completely different chemical structure, does not have an imidazole ring, and belongs to amide local anesthetics. Simultaneous quantitative determination by HPLC method for these analytes is feasible however there are certain challenges primarily due to the large difference in polarity: as metronidazole, being relatively polar (elutes first), lidocaine, being moderately lipophilic, while miconazole and clotrimazole, highly lipophilic, (elutes late, often requiring a gradient). Gradient elution may compromise method robustness and reproducibility, particularly in routine quality control environments where simplicity and repeatability are critical, so these methods are long lasting and cost effective. Another major challenge is related to the ionizable nature of the analytes. All the four compounds contain functional groups capable of protonation or deprotonation depending on mobile-phase pH. Inadequate pH control can result in variable retention behavior, peak tailing, and strong secondary interactions with residual silanol groups or metal impurities in the stationary phase. These effects are especially pronounced for basic compounds such as lidocaine and imidazole derivatives, leading to asymmetric peak shapes and reduced resolution. Furthermore, achieving adequate selectivity between structurally related imidazole derivatives (clotrimazole and miconazole) while maintaining reasonable retention for the polar metronidazole requires careful optimization of stationary phase chemistry, mobile-phase composition, and acidic modifiers [3–6, 29]. Therefore, the development of aт accurate HPLC method capable of simultaneously separating all the four analytes and one UV-absorbing nitrate residue from miconazole treated as impurity within a single run requires a balanced optimization of selectivity, efficiency, analysis time, and method robustness. In the literature, the determination of certain analytes has been described however not all four together. Metronidazole, lidocaine, and miconazole were simultaneous determined using a RP-HPLC method in semi-solid (intravaginal) formulations: Zorbax RX-C8 column (250 × 4.6 mm, 5 µm), detection at 215 nm, gradient with mobile phase A (0.1% H₃PO₄ + 0.1% HClO₄) and B (ACN), flow rate 1.2 mL/min; typical retention times are also given (~ 3.2, 5.5, 9.3 min, respectively) [25]. In this method an early elusion of metronidazole, about ~ 1 min., and much later one for clotrimazole, about ~ 11 min., was noticed) [26]. For metronidazole and miconazole, there is a classical RP-HPLC approach on C18 with MeOH:water 40:60, 1.0 mL/min, detection at 254 nm [27].

While developing a HPLC method for the simultaneous determination of four analytes, various approaches were tested:

• the choice of a chromatographic column (C18, CN, phenyl);

• the choice of mobile phase (organic component (ACN, MeOH + diluted acid solution (H₃PO₄, TFA, HCOOH))—the acidic aqueous component (pH ~ 2–3) usually helps stabilize peaks, control lidocaine ionization, and improve reproducibility;

• detection—in the scientific literature, 210–215 nm was used for combinations;

• sample preparation (for creams/gels/suppositories) usually requires an organic extractant (MeOH, ACN or a mixture), sonication, volume adjustment, centrifugation/0.45 μm filtration.

The first tested column was LiChrosphere CN column, (250 × 4 mm, 5 µm) and the same one type LiChrospher CN (125 × 4 mm, 5um) column. Results are shown in Figs. 1 and 2.

Fig. 1.

Chromatogram of separated four API components on LiChrosphere 100 CN 250 × 4 mm 5 µm column with mobile phase composed of MeOH:ACN:0.05% TFA (35:35:30) at flow rate 1 mL/min, UV = 215 nm, Column temp. 35 °C (the right side of the figure presents overlaid UV-spectra of all four API peaks and nitrates residue from miconazole, with their retention times for identification)

Fig. 2.

The linearity of the method using shorter LiChrosphere 100 CN 125 × 4 mm 5 µm column, under same parameters amentioned before, Chromatogram of separated four API components on LiChrosphere 100 CN 250 × 4 mm 5 µm column with mobile phase composed of MeOH:ACN:0.05% TFA (35:35:30) at flow rate 1 mL/min, UV = 215 nm, Column temp. 35 °C, with same elution order: nitrate, metronidazole, lidocaine, clotrimazole and miconazole

From Figs. 1 and 2, which illustrate the HPLC analysis, the results confirm the validity of the method. All five peaks are well resolved and exhibit good symmetry, with separation achieved within a short run time. Considering that the column used could 250 or 125 mm long and packed with 5 µm particles, yielding approximately 12,000–13,000 number of theoretical plates per meter (NTP) for longer 250 mm and so with half values of NTP for shorter of 125 mm length column. These columns represent a good choice from a selectivity standpoint for the simultaneous determination of one or more of the mentioned analytes. Both column have satisfying Resolutions in range 4.3–22.5 with 250 mm and within 3.1–15.8, about 1.42 times reduced to 125 mm halved column length. After this, Peak symmetries are in range 1.1–1.5 for both column sizes, which confirms the method separation parameters. These findings indicate that selectivity is the primary chromatographic achievement, despite the relatively modest overall column performance parameters such as NTP, particle size, end-capping, and base-deactivation treatment. The LiChrosphere CN column, which is not end-capped or base-deactivated and has lower efficiency (NTP) under acidic conditions, nevertheless produced symmetric peaks. This behavior may be attributed to ion-pairing between TFA and the nitrogen atoms of imidazole-based molecules, thereby reducing their charge and minimizing secondary interactions with silanol groups and embedded metal cations in the silica matrix. The same experimental conditions were subsequently applied in linearity testing using a column of twice the length, packed with identical matrix particles (LiChrosphere 250 × 4 mm, 5 µm), and the following results were obtained (Fig. 3). Excessive doubled number of theoretical places as result of twice longer column is noticeable, with slight increasing of peak tailing due to longer retaining.

Fig. 3.

The linearity of the method using shorter LiChrosphere 100 CN 250 × 4 mm 5 µm column, under same method parameters as that on previous Fig. 1, Chromatogram of separated four API components on LiChrosphere 100 CN 250 × 4 mm 5 µm column with mobile phase composed of MeOH:ACN:0.05% TFA (35:35:30) at flow rate 1 mL/min, UV = 215 nm, Column temp. 35 °C, with same elution order: nitrate, metronidazole, lidocaine, clotrimazole and miconazole

The chromatogram presented in Fig. 3 confirms the superiority of the LiChrosphere CN column in separation selectivity for four API analytes, providing well-spaced retention times and comfortable peak positioning. This feature is particularly advantageous for combined pharmaceutical formulations, where additional peaks may arise from newly introduced analytes, degradation products, or other formulation-related components. A direct comparison of chromatograms obtained under identical experimental conditions using columns packed with the same stationary phase but differing only in column length is shown in Fig. 4.

Fig. 4.

Overlaid chromatograms comparing the separation performance of identically packed HPLC columns (LiChrosphere 100 CN, 5 µm) with lengths of 125 and 250 mm are shown on the left, while the corresponding overlaid UV spectra are presented on the right

These separations performed on cyano-bonded columns showed very interesting results for the separation and determination of mixtures containing analytes with markedly different chemical properties, such as hydrophobicity and polarity. When comparing the characteristics of metronidazole, lidocaine, and the highly lipophilic compounds clotrimazole and miconazole, concerns arise regarding their isocratic separation on alkyl-bonded reversed-phase silica columns. In such systems, the very long retention times of the most hydrophobic analytes would excessively extend the overall analysis time. A similar situation would be encountered when using gradient elution on alkyl-bonded C8 or C18 columns: although effective, such methods are often time-consuming and cost-inefficient. These type of column are very applicative in these research and analytics, since depending of analytical task, we can double the separation power of method only with change of column length. If we need routine quantitative high-throughput analyses with shorter run last, we will choose 125 mm column, in stability studies or in presence of additional peaks of APIs or degradation products, related compounds, we will chose more powerful 250 mm column.

Further experimental investigations were performed using a Waters Spherisorb CN column (250 × 4.6 mm, 5 µm), in which the content of organic solvent and diluted acids was varied. Two different acids were tested: 0.04% H₃PO₄ and 0.1% TFA (Figs. 5, 6, 7, 8 and 9). This column exhibited a higher NTP. The same sequence of elution and retention times was observed, with increased efficiency (NTP) compared with the previously used LiChrosphere CN column.

Fig. 5.

Chromatogram gained on Spherisorb CN column 250 × 4.6 mm 5 µm and mobile phase 0.04% v.v H₃PO₄: MeOH: ACN = 40:30:30, and 1.2 mL/min, with elution order of elution: imp. nitrate, metronidazole, lidocaine, clotrimazole, and miconazole

Fig. 6.

Influence of level of acidification with chromatograms gained with Waters Spherisorb CN column 250 × 4.6 mm 5 µm and mobile phase of 0.1% v/v TFA:MeOH:ACN = 40:30:30 and 35:30:35, and at 1.2 mL/min, with same elution order of elution: nitrate, metronidazole, clotrimazole, miconazole and lidocaine in upper chromatogram, and changed last 2 peaks elution order to: nitrate, metronidazole, clotrimazole, miconazole and lidocaine in lower chromatogram

Fig. 7.

Chromatogram with Waters Spherisorb CN 250 × 4.6 mm 5 µm with 40:30:30–0.1%TFA composed mobile phase and the overlaid UV-spectra confirmation on the right side

Fig. 8.

Comparison and influence of presence of H₃PO₄ in mobile phase on resolution (the top chromatogram with 45:25:30 – a.b.c = a-0.05% TFA, bellow-a- 0.1% TFA)

Fig. 9.

Analysis of Comb stds and two different suppositories products, one with two API and second with three API, with one universal method which is described by using Waters Spherisorb CN column 250 × 4.6 mm 5 µm particles and mobile phase constituents 0.05% TFA: MeOH: ACN = 45: 25: 30

This column showed interesting sensitivity to small changes in mobile phase composition changes, which is presented in following Fig. 6 and following Fig. 7.

We can conclude that this second type and vendor of cyano-bonded column is very powerful and applicative too in separation and determination of four API analytes with imidazole based structures in different dosage forms. These facts are visible and presented in the Fig. 9.

The third other tested CN-bonded column was Agilent Zorbax SB CN 250 × 4,6 mm 5 µm particles, and using of this column appeared quite different characteristics from previous used. Firs retention times were shorter, NTP was highest with and narrowest peaks, and most interesting was change of elution order of analytes, appearing peaks of nitrates as last one of baseline.

This column provided the fastest separation of the four analytes, exhibiting characteristics of a high-throughput analytical method suitable for rapid routine control analysis (Fig. 10); however, its application may be limited by the close elution of components, which increases the risk of co-elution with structurally similar compounds in the same elution regions. The other very interesting feature of this column is behavior like normal-phase separated component in opposite appearance from classic reversed phase order, as chromatogram on the Fig. 10 presents. The most polar and most hydrophilic components are eluting last with highest retention times for metronidazole and nitrate anion as last eluting appearance. This column can be used for efficient high-throughput analyses with shortest run lasting time, but in samples where other components do not co-elute in vicinity of these 5 peaks.

Fig. 10.

Chromatogram obtained with Agilent Zorbax SB CN 250 × 4.6 mm 5 µm column, with 30:35:35 = 0.1% H₃PO₄: MeOH: ACN (composition of mobile phase), flow rate at 1.2 mL/min, Column temp. 42 °C and Inj Vol = 2 µL. The new moment is reversed appearance of peak elution retention times, metronidazole and nitrate as last eluting

Zorbax SB Phenyl (150 × 4.6 mm 3.5 μm) column was also investigated and unexpectedly excellent results were obtained, characterized by good separation and symmetric peaks for four API analytes as well as the nitrate impurity of miconazole. For comparison, Figs. 11 and 12 present chromatograms obtained on this column using a mobile phase consisting of ACN, MeOH, and 0.05% H₃PO₄ (35:30:35), at column temperatures of 42 °C (Fig. 11) and 45 °C (Fig. 12), with a flow rate of 1.0 mL/min.

Fig. 11.

Chromatogram obtained with Agilent Zorbax SB Phenyl (150 × 4.6 mm 3.5 μm) column, with ACN, MeOH, and 0.05% H₃PO₄ (35:30:35) (composition of mobile phase), flow rate at 1.0 mL/min, T = 42 °C

Fig. 12.

Chromatogram obtained with Agilent Zorbax SB Phenyl (150 × 4.6 mm 3.5 μm) column, with ACN, MeOH, and 0.05% H₃PO₄ (35:30:35) (composition of mobile phase), flow rate at 1.0 mL/min, T = 45 °C

The elution order on the Phenyl-based column is the same as the first 2 CN-columns, LiChrosphere CN and Spherisorb CN, corresponding to characteristic’s of more hydrophobic and low polar interaction based columns, presumably alkyl-chain based C18 and C8, followed by Phenyl-based columns, which is in the middle between alkyl-RP and CN-polar interaction columns.

All approaches proposed by us for column selection and for optimizing the composition and ratios of the mobile-phase components are effective, meet chromatographic system suitability criteria, and can be selected according to the specific analytical objectives (e.g., whether an ultra-fast analysis is required and which combination of columns and solvents is most appropriate). These outcomes were achieved as a result of numerous labor-intensive experiments aimed at identifying optimal separation conditions for four API analytes and one impurity (five components in total). Such tasks are inherently complex and require thorough knowledge of chromatographic theory as well as substantial practical experience.

Validation of analytical method

Methods with all four columns were validated in accordance with ICH requirements [30]. Below are the validation results on the chromatographic column LiChrosphere CN.

Linearity

Linearity was evaluated by constructing calibration curves for clotrimazole, metronidazole, miconazole, and lidocaine at six concentration levels spanning the studied ranges. Peak area was plotted against the corresponding analyte concentration, and linear regression analysis produced correlation coefficients of 0.9991, 0.9996, 0.9991, and 0.9992 for clotrimazole, metronidazole, miconazole, and lidocaine, respectively. A linear response was observed over the concentration range of 20–120 µg/mL for clotrimazole, miconazole, and lidocaine, and 80–500 µg/mL for metronidazole. The calibration curve parameters, including the standard deviations of the slope (Sb) and intercept (Sa), are summarized in Table 1.

Table 1.

Validation parameters and results obtained by the proposed RP-HPLC method

Item	Clotrimazole	Metronidazole	Miconazole	Lidocaine
Range of linearity, μg mL−1	20–120	80–500	20–120	20–120
Regression equation	y = 3.8293x-12.295	y = 0.5055 x-4.6407	y = 2.8138x-10.969	y = 4.4849x-19.553
Regression coefficient (r2)	0.9991	0.9996	0.9991	0.9992
LOD (3.3σ/S) (μg mL−1)	1.72	7.75	1.11	2.30
LOQ (10σ/S) (μg mL−1)	5.22	22.89	3.36	6.97
Standard error of estimate	10.2091	20.7439	20.8450	15.9403
Intra-day RSD (%)	0.092–0.240	0.078–0.178	0.059–0.163	0.078–0.590
Inter-day RSD (%)	0.090–0.463	0.024–0.089	0.040–0.205	0.043–0.334
Drug in bulk	101.025 ± 0.637	100.302 ± 0.860	99.595 ± 0.803	99.441 ± 0.784
Drug in dosage form	100.553 ± 0.593	100.605 ± 0.649	100.830 ± 0.593	100.603 ± 0.528
Drug added	99.934 ± 0.674	100.849 ± 0.694	100.054 ± 0.395	100.514 ± 0.6749

Accuracy

The accuracy of the HPLC method reflects its ability to produce results that are close to the true value. In the present study, accuracy was evaluated by determining percent recoveries at six different concentration levels, each analyzed in triplicate. As summarized in Table 1, the recoveries for all drugs were greater than 98%.

Limit of detection and limit of quantitation

In accordance with ICH guidelines, the limit of detection (LOD) and limit of quantitation (LOQ) were estimated based on the slope of the calibration curve and the standard deviation of the response. The obtained results are presented in Table 1.

Precision

Precision was evaluated in terms of repeatability (intra-day) and intermediate precision (inter-day). Intra-day precision was assessed by analyzing six replicates at three concentration levels within the same day, whereas inter-day precision was evaluated over three consecutive days. As shown in Table 1, the calculated %RSD values for all analytes were below 2%, demonstrating that the method provides consistent and reliable results under normal operating conditions.

System suitability tests

The system suitability features were described NTP, resolution factor (R), tailing factor (T). Table 2 reports the test results for the developed approach.

Table 2.

System suitability tests for the developed RP-HPLC method

Parameter	Clotrimazole	Metronidazole	Miconazole	Lidocaine	Reference value
NTP	3864	3002	5636	4506	The higher the value, the more efficient the column is
R	2.537	–	2.776	12.491	> 2
T	1.865	1.753	1.264	1.688	≤ 2

Specificity

Specificity is defined as the ability of an analytical method to accurately measure the analyte in the presence of components that may be expected to be present in the sample matrix. In this study, specificity was evaluated to ensure that formulation excipients did not interfere with the analysis (Fig. 13). It was assessed by analyzing each drug individually in its dosage form, and no interference from excipients was observed. Furthermore, as shown in Table 3, specificity was confirmed by analyzing laboratory-prepared mixtures containing different proportions of the investigated drugs.

Fig. 13.

Specificity chromatogram

Table 3.

Determination of clotrimazole, metronidazole, miconazole, and lidocaine in laboratory prepared mixtures using the proposed RP-HPLC method

Taken (µg/ml)				Found (µg/ml)				Recovery %
Clotrimazole	Metronidazole	Miconazole	Lidocaine	Clotrimazole	Metronidazole	Miconazole	Lidocaine	Clotrimazole	Metronidazole	Miconazole	Lidocaine
20	80	20	20	20.134	79.591	20.189	19.849	100.67	99.489	100.945	99.245
40	164	40	40	39.947	164.239	39.329	40.565	99.867	100.146	98.323	101.412
60	248	60	60	60.127	247.483	60.394	60.132	100.212	99.791	100.657	100.220
80	332	80	80	79.385	332.189	80.045	80.243	99.231	100.057	100.056	100.304
100	416	100	100	100.674	415.478	100.389	99.495	100.674	99.875	100.389	99.495
120	500	120	120	121.392	499.834	119.843	120.483	101.160	99.667	99.869	100.403
Mean								100.302	99.837	100.040	100.180
SD								0.68	0.24	0.93	0.76
SE								0.30	0.16	0.04	0.18

Integrated sustainability evaluation of the proposed method

Evaluating the sustainability of analytical methods has become essential for understanding their efficiency, safety, environmental impact, and economic feasibility [31–35]. While Green Analytical Chemistry focus on environmental friendliness and analytical performance, respectively, the concept of Blue Analytical Chemistry highlights the practical applicability of developed methods. In this study, the proposed method was assessed using established evaluation tools: AGREE and MoGAPI for greenness, BAGI for blueness.

Analytical greenness tool (AGREE)

AGREE is a straightforward tool introduced in 2020 to evaluate the environmental impact of analytical methods. The assessment is presented as a pictogram divided into twelve sectors, each representing a principle of Green Analytical Chemistry (GAC). Each sector is scored on a scale from 0 to 1, where 1 indicates a fully green practice and 0 indicates a non-green practice. The color of each sector ranges from dark green to red, and an overall score displayed at the center of the pictogram reflects the method’s overall greenness. As shown in Fig. 14a, the proposed method achieved an AGREE score of 0.68. Although some reported methods may exhibit shorter run times or higher AGREE scores, these methods typically determine only one or two drugs, whereas the proposed method enables the simultaneous determination of four drugs.

Fig. 14.

Sustainability evaluation using: (a) AGREE, (b) MoGAPI, (c) BAGI

Modified green analytical procedure index (MoGAPI)

MoGAPI, developed in 2024, is an advanced tool for evaluating method greenness that combines the color-coded pictogram of the original GAPI with a quantitative overall greenness score. The MoGAPI pictogram is composed of interconnected pentagons, each reflecting the environmental impact of specific analytical parameters, where green indicates low impact, yellow denotes moderate impact, and red signifies high environmental impact. As illustrated in Fig. 14b, the MoGAPI pictogram of the proposed method shows red fields corresponding to offline operation, macroextraction procedures, the use of acetonitrile in the mobile phase, and the absence of a waste treatment step. Nevertheless, the predominance of green and yellow fields, along with a greenness score of 76, indicates a favorable environmental profile.

Blue applicability grade index (BAGI)

BAGI is a recently introduced tool used to assess the practical applicability of analytical methods based on ten criteria, including the nature of the analysis, multi-analyte capability, instrumentation, sample handling capacity, sample preparation, sample throughput (number of samples analyzed per hour), reagents and materials, preconcentration requirements, degree of automation, and sample volume. The BAGI output is displayed as a schematic diagram consisting of graded fields, where darker blue regions indicate higher applicability and are accompanied by a numerical score. As illustrated in Fig. 14c, the proposed method achieved an overall BAGI score of 67.5, indicating good practical applicability.

Conclusion

This study presents an efficient isocratic RP-HPLC method developed and optimized for the simultaneous quantification of clotrimazole, miconazole, metronidazole, and lidocaine. The proposed method achieved excellent chromatographic separation and demonstrated satisfactory system suitability. All validation parameters were successfully met in accordance with ICH guidelines, confirming the reliability and accuracy of the method. The importance of this research lies in providing a unified analytical approach for the simultaneous determination of multiple active pharmaceutical ingredients, which simplifies quality control procedures and reduces analysis time and cost. The novelty of the method is reflected in the simultaneous analysis of these four compounds using a single isocratic run, combined with a comprehensive evaluation of sustainability through greenness, blueness, and whiteness metrics. Owing to its simplicity, cost-effectiveness, high reproducibility, and environmentally conscious design, the developed method represents a valuable analytical tool for routine quality control, particularly in pharmaceutical settings dealing with multicomponent formulations, and is well suited for routine analysis and therapeutic monitoring.

Abbreviations

HPLC

High-performance liquid chromatography

Eur. Ph.

European Pharmacopeia

QC

Quality control

API

Active pharmaceutical ingredient

MeOH

Methanol

ACN

Acetonitrile

RC

Regenerated cellulose

NTP

Number of the theoretical plates

Tf

Tailing factor

Rs

Resolution

Rt

Retention time

LOD

Limits of detection

LOQ

Limits of quantification

Author contributions

All authors have read and agreed to the published version of the manuscript. Andrii Zhuravel: Investigation, Supervision, Methodology, Resources, Formal analysis, Data curation, Writing—original draft, Writing and review and editing; Volodymyr Kopylevych, Liliya Logoyda: Supervision, Formal analysis, Writing and review and editing; Marjan Piponski, Mamdouh R. Rezk, Antonija Beneta, Ivan Smachylo, Nadiya Zarivna, Olha Poliak: Resources, Formal analysis, Data curation, Writing—review and editing.

Funding

Non-funding.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.