Abstract
A fast reliable micellar electrokinetic methodology was investigated for the concurrent quantitation of six antimicrobial and anti-inflammatory drugs, namely, ciprofloxacin, dexamethasone, metronidazole, ornidazole, spiramycin and tinidazole. The method has the merits of rapidity, precision, and sensitivity. The separation was carried out in less than 7 min by applying a basic background electrolyte consisting of 25 mM disodium tetraborate buffer, pH 9 containing 50 mM SDS at 25 kV using photodiode array detector at 230 and 315 nm. The internal standard used during analysis was cromolyn sodium and validation was carried out following ICH guidelines. The proposed method showed linear response over the range from 0.5 to 10.0 μg mL−1 reaching limits of detection and limits of quantitation in the ranges of 0.09–0.2 μg mL−1 and 0. 27–0.6 respectively. The method's greenness was estimated using the GAPI tool where excellent greenness was concluded. Co-formulated or single-ingredient commercial preparations were investigated and the results were statistically evaluated.
1 Introduction
Ciprofloxacin (CIP) is a broad spectrum antibacterial agent belonging to the group of fluoroquinolones [1]. CIP is used with an antimicrobial medication that kills anaerobes, like metronidazole (MNZ), to treat mixed aerobic/anaerobic infections [2]. On the other hand, a combination of CIP and ornidazole (ORZ) is used in the treatment of bacterial & parasitic infections. It is used to treat diarrhoea, gynaecological and pelvic infections. It also helps treat mixed infections of teeth and gums. CIP works by preventing the bacterial cells from dividing and repairing, thereby killing the bacteria. ORZ kills parasites and anaerobic bacteria that cause infections by damaging their DNA [3]. At the same time, combined CIP and tinidazole (TNZ) was found to be effective in the cases of pelvic inflammatory disease [4] while, CIP and dexamethasone (DXM) otic drops are used to treat outer ear infections in adults and children and acute (suddenly occurring) middle ear infections in children with ear tubes. The combination of CIP and DXM works by killing the bacteria that cause infections and reducing swelling in the ear. Additional uses have been described in children with Acute Otitis Media and Otorrhea (AOMT) [5], topical otic therapy with CIP/DXM is superior to CIP alone and leads in a faster clinical remission, furthermore a combination dosage form of spiramycin (SPI) and metronidazole (MNZ) was found to be effective in the treatment of active periodontitis [6]. This combination is beneficial in reducing brain cysts caused by toxoplasma gondii [7]. As different combinations present in the market containing two or more of the six cited drugs, we aimed to develop a fast reliable capillary electromigration separation method for the quantitation of these antimicrobial and anti-inflammatory drugs in their different dosage forms, based on MEKC. This will help in quality control testing without the need for sample preparation and costly solvents. Additionally, simultaneous estimation plays a very important role in the pharmaceutical analysis as it is very feasible and time saving in quality control laboratories.
Figure 1 illustrates the chemical structure of the studied analytes. Various methods have been developed for the analysis of these compounds in the literature. A good guide for the work reported for CIP up to 2018 is found in the critical review written by Kawas et al. [8]. Another review article covering the reported methods for ORZ up to 2014 was prepared by Zameeruddin et al. [9]. Regarding TNZ, Sebastian [10] reviewed its literature up to 2017. The recent articles concerning the analysis of CIP include spectrophotometric [11, 12], High performance liquid chromatography (HPLC) [13, 14] and capillary electrophoresis (CE) [15, 16] methods. Regarding DXM, the recent methods include spectrophotometric [17], TLC [18], HPLC [19, 20] and CE [21] methods. For MNZ, the reported methods include spectrophotometric [22, 23] electrochemical [24, 25], TLC [26, 27] HPLC [28, 29] and CE [30, 31] methods. Determination of ORZ was carried out using spectrophotometric [32, 33] and HPLC [34, 35] methods. The literature for SPI determination includes spectrophotometric [36], HPLC [37, 38], UHPLC [39, 40] and CE [41] methods. As for TNZ, the recent methods include spectrophotometry [42, 43], electrochemical determination [44], TLC [45] and HPLC [46, 47].
Chemical structure of: (A) Ciprofloxacin HCL; (B) Dexamethasone; (C) Metronidazole; (D) Ornidazole; (E) Spiramycin; (F) Tinidazole
Citation: Acta Chromatographica 35, 3; 10.1556/1326.2022.01057
Micellar Electrokinetic Chromatography (MEKC) is a technique that relied on adding surfactants to the background electrolytes [48]. This technique permits the simultaneous separation of both neutral and charged species. The surfactant is added to the background electrolyte (BGE) above its critical micelle concentration (CMC) where individual surfactant molecules aggregate to form micelles. The micelles are dynamic structures with hydrophobic tails positioned in the center away from the hydrophilic buffer and charged heads located outside and towards the aqueous buffer. As the micelles are charged, they act as a pseudo-stationary phase for the analytes and the partitioning of the analytes occur between the moving micellar and aqueous phases which affects the separation process. Micelles and solutes interactions are electrostatic and/or hydrophobic in nature. During electrophoresis micelles also show a differential migration due to their effective mobility. For a neutral species, the interactions are essentially hydrophobic whereas for a charged species, they are a mixture of electrostatic and hydrophobic interactions [49]. As polarity plays an important role in the analysis of pharmaceuticals, the introduction of MEKC resulted in versatility of applications in this area [50–52].
The Society of Analytical Chemistry is recently following the applications of Eco-friendly methods that exclude or reduce toxic and caustic waste [53]. Assessment of the greenness of analytical methods is now an important issue in the development stage. The small scales in which MEKC separations are performed make this family of techniques a greener alternative to chromatography. These approaches consume less amounts of solvents and samples. Using micellar biodegradable system is an additional merit. Among the new methods used for greenness evaluation is GAPI [54]. It is superior to other green assessment tools because it is based on the evaluation of the green character of an entire analytical methodology, from sample collection to final determination. We, therefore, evaluated the greenness using GAPI method.
2 Materials and methods
2.1 Materials
Pure CIP, TNZ and SPI were supplied by the National Organization of Drug Control and Research (NODCAR), Cairo, Egypt.
Pure ORZ was provided by Pharonia Pharmaceuticals (Pharo Pharma), Borg EL-ARAB, Alexandria, Egypt.
Pure MNZ was provided by Amriya Pharmaceutical Industries, Alexandria, Egypt.
Pure DXM was provided by Egyptian International Pharmaceutical Industries Company (EIPICO), 10th of Ramadan City, Egypt.
Cromolyn Sodium was provided by Sigma Pharmaceutical Company, 6th October City, Egypt, used as the internal standard (IS).
Pharmaceutical preparations were bought from the local Egyptian market. These include
Ciprobay® tablets; containing 500 mg of CIP, product of Hikma Pharma S.A.E. Egypt.
Ornidaz® tablets; containing 500 mg of ORZ, product of Chemipharm Pharmaceutical Industries S.A.E. 6th October City, Egypt.
Protozole tablets; containing 500 mg of TNZ, product of Medical Union Pharmaceuticals, Ismaelia, Egypt.
Flagyl ® tablets; containing 500 mg of MNZ, product of Sanofi Aventis. Cairo, Egypt.
Dexamethasone-MUP ampoules Batch No.20246, each ampoule contains 8 mg dexamethasone phosphate produced by Medical Union Pharmaceuticals, Abu-Sultan, Ismailia, Egypt.
Spirex® tablets; product of Medical Union Pharmaceuticals, Ismaelia, Egypt.
Tinifloxacin tablets, containing 500 mg of CIP and 600 mg of TNZ, produced by Organopharma. Cairo, Egypt.
Ciprodiazole tablets, contain 500 mg CIP and 500 mg MNZ/tablet, by Minapharm for Pharmaceuticals and Chemical Industries 10th of Ramadan City- Egypt.
Co-formulated tablets Ciplox-OZ [55] were prepared in laboratory by mixing 10 mg CIP and 10 mg ORZ fine powder together. The powder was compacted into tablets with common diluents and fillers.
Spirazole forte tablet, containing 1.5 M.I.U spiramycin and 250 mg metronidazole; produced by Pharonia Pharmaceuticals (Pharo Pharma), Borg EL-ARAB, Alexandria, Egypt.
Zonacip otic drops, containing 3 mg CIP and 1 mg DXM/mL; produced by Pharonia Pharmaceuticals (Pharo Pharma), Borg EL-ARAB, Alexandria, Egypt.
2.2 Reagents
Analytical grade NaH2PO4, Na2B4O7.10 H2O (borax), H3PO4 and NaOH were from Merck, Darmstadt, Germany.
Sodium dodecyl sulfate (SDS) was bought from Fluka, Buchs, Switzerland.
0.45 μm Syringe filters (Minisart RC25) were bought from Sartorius-Stedim (Gottingen, Germany) were used for filtration of samples and BGE.
Deionized water was used throughout the study.
2.3 Instruments
➢ Agilent 7100 Capillary Electrophoresis system (Agilent Technologies, Waldbronn, Germany) with a diode-array detector was used. Data were recorded with Agilent Open LAB CDS software. Fused-silica capillaries (50 μm I.D.) were from Agilent Technologies (Waldbronn, Germany) with a total length of 48.8 cm and effective length: 40.5 cm the generated electric current was around 35 μA under the optimized conditions,
➢ For pH measurements, a Consort NV P-901 pH–Meter (Belgium) was used.
➢ Ultrasonic bath BHA-180 T (Abbotta, USA).
2.4 Preparation of background electrolyte
Borate buffer stock (100 mM, pH 9.0) was prepared by dissolving 3.714 g of disodium tetraborate (Na2B4O7.10H2O) in 100 mL of deionized water and the final pH was adjusted to pH 9.0 with 0.1 M NaOH. Accurately weighted 5.778 g SDS was dissolved in 100 mL of deionized water to obtain a stock SDS solution (200 mM) and the solution was kept in the refrigerator and used within one week. These stock solutions used for the preparation of the final optimized BGE which is a 20 mM borate buffer with pH of 9. 0 and 50 mM SDS. The BGE was degassed in an ultrasonic bath for 10 min after being filtered using a 0.45 mm syringe filter.
2.5 Standard solutions
Freshly prepared stock solutions containing 1.0 mg mL−1 of each of TNZ, MNZ, ORZ, CIP, DXM, SPM and Cromolyn sodium (I.S) were prepared separately in deionized water. Working solutions were prepared by further dilution with the same solvent to obtain concentration of 0.1 mg mL−1.
2.6 Electrophoretic procedure
➢ For the first usage, the capillary was conditioned by flushing with 1.0 M NaOH for 30 min, then with water for 15 min, followed by 5 min of drying by purging air through the capillary.
➢ The capillary was rinsed for 30 min with 0.2 M NaOH, 10 min with water, and then BGE for 30 min at the start of each working day.
➢ To keep optimal run-to-run injection repeatability, the capillary was preconditioned with 0.2 M NaOH for 2 min, water for 1 min and BGE for 3 min before each injection. .
➢ Photodiode array (PDA) detector was set at 230 and 315 nm with a bandwidth of 10 nm.
➢ Samples were introduced at hydrodynamic pressure of 50 mbar for 10 s,
➢ The temperature of the capillary was kept constant at 25 °C and a voltage of 25 kV was applied (positive polarity).
3 Procedures
3.1 Construction of Calibration Graphs
In a set of 10-mL volumetric flasks, aliquots of the working standard solutions of the six drugs (0.1 mg mL−1) were transferred and a constant volume of 10 μL cromolyn sodium stock solution (1.0 mg mL−1) was added. The flasks were completed to volume with deionized water to reach final concentrations in the range of 0.5–5.0 μg mL−1 for TNZ and ORZ, 1.0–10.0 μg mL−1 for MNZ, CIP. The peaks of the corresponding drugs were detected at 315 nm while DXM and SPI were detected at 230 nm for treplicate and average responses were calculated. The samples were then analyzed under the above optimum conditions. The corrected peak area ratio was plotted accordingly versus the final concentration of each drug in μg mL−1 and the corresponding regression equation was derived.
3.2 Pharmaceutical applications
3.2.1 For single-component tablets
Ten Ciprobay®, Ornidaz®, Flagyl®, Protozole or Spirex ® tablets were weighed and finely powdered. An amount equivalent to 10.0 mg of each drug was transferred in to separate 100-mL volumetric flasks and about 50 mL of deionized water was added. The flasks were then sonicated for 20 min, completed with deionized water to obtain a solution containing 0.1 mg mL−1 for each drug. The solutions were then filtered through a syringe filter.
3.2.2 For DXM in injections
Into a 100-mL volumetric flask, accurately measured volumes of freshly mixed five Dexamethasone ® ampoules equivalent to 10 mg DXM were transferred. About 75.0 mL of deionized water were added, mixed, and completed to obtain a solution containing 0.1 mg mL−1 DXM.
3.2.3 For multi-component tablets
Ten tablets of Ciplox-OZ [55], Ciprodiazole, Tinifloxacin and Spirazole forte ® tablets were grinded then transferred into four separate 100-mL volumetric flasks and about 50.0 mL of deionized water were added and mixed well then sonicated for 20 min. Volumes were completed to the mark and filtered.
3.2.4 Zonacip otic drops
The contents of five Zonacip otic bottles were mixed and a volume of the freshly mixed suspension equivalent to 30 mg of CIP and 10 mg of DXM was transferred to 100-mL volumetric flask. The flask was completed to the mark with deionized water, then filtered using a syringe filter.
As illustrated under “Construction of Calibration Graphs,” further dilution with deionized water was made as suitable for each preparation. Each analyte's corrected peak area ratio (analyte/I.S.) was calculated and the concentration was estimated using the corresponding regression equation.
4 Results and discussion
Various combinations in dosage forms are present in the pharmaceutical market. These multi component formulations need new methods for their analysis. There is increasing interest in the use of capillary electrophoresis for applications in analytical and biochemical fields. Two problems were faced while attempting to separate the studied analytes: The first problem is the different pka range between the analytes. The second one is the structural similarity of the three studied nitroimidazole, which need to be separated by high resolution power. MEKC has thought to be a promising method for separating charged and neutral molecules with high resolving power, small sample volumes, and less time compared with HPLC. The designated method permitted the separation of the six studied drugs in a single run (less than 7 min) with good resolution. Figure 2A and B show typical electropherograms for the studied drugs under the described conditions at 315 and 230 nm. The residence times for TNZ, MNZ, ORZ, CIP, DXM and SPI were 2.5, 2.6, 2.8, 3.6, 4.0 and 6.8 min, respectively.
(A) Electropherogram for the studied analytes using the optimized experimental conditions at 315 nm; (analyte concentration: 2.0 µg mL−1 for TNZ and ORZ, 5.0 µg mL−1 for MNZ and CIP and 1.0 µg mL−1 for Cromolyn Sodium as I.S.; A: TNZ (2.5 min), B: MNZ (2.6 min), C: ORZ (2.8 min), D: CIP (3.6 min) and I.S: (4.2 min). (B) Electropherogram for the studied analytes using the optimized experimental conditions at 230 nm; (analyte concentration: 5.0 μg mL−1 for DXM and SPI; D: CIP (3.6 min), E: DXM (4.0 min), I.S: (4.2 min), F: SPI (6.8 min)
Citation: Acta Chromatographica 35, 3; 10.1556/1326.2022.01057
4.1 Optimization of capillary electrophoretic conditions
All parameters influencing the efficiency and selectivity of the proposed method were studied and optimized.
4.2 Detection wavelength
Depending on the absorbance properties of each compound Diode array detector was set at different wavelengths. At wavelengths of 200, 220, 230, 270, and 315 nm, different electropherograms were run. TNZ, MNZ, ORZ, and CIP electropherograms were found to be the best at 315 nm while the best electropherograms were obtained at 230 nm for DXM and SPI, so the two wavelengths were used for detection to enhance the sensitivity the method.
4.3 Rinsing of the capillary between two runs
Alkaline conditioning to remove adsorbates and refresh the surface by deprotonation of the silanol groups is most commonly employed.
In preliminary studies the capillary was rinsed with 0.1 M NaOH for one minute, deionized-water for one minute and buffer solution for three minutes. The steps were performed at a pressure of 50 mbar. Also 0.1 M NaOH for 2 min, water for 1 min and BGE for 3 min was tried but a delay in the migration time was noted. This led to the use of 0.2 M NaOH. Different durations of the washing steps were investigated and 0.2 M NaOH for 2 min, water for 1 min and BGE for 3 min was the best rinsing procedure that maintained appropriate injection repeatability from run to run.
4.4 Effect of pH
It is an important notice to state that the pka values of TNZ, MEZ, ORZ, CIP, DXM and SPI are 4.7,2.62,2.4,6.09,1.89 and 8.0 respectively [56], so alkaline pH would be an excellent choice for separation. The selection of alkaline pH instead of neutral or acidic was also relied on the fact that the alkaline medium holds the capillary tube's inner silica ionized [57] thus reducing the migration time.
The effect of pH was examined over the pH range 6–10, using 20 mM buffer solutions prepared at various pH values. As the pH increased, the migration times were shown to decrease. Lower pH values resulted in an increase in the migration time with accompanying peak broadening of TNZ which is co-eluted with solvent front. The optimum pH was 9.0 as it resulted in the highest efficiency, well resolved peaks, best peak shape, and sensitivity (Fig. S1).
4.5 Buffer type and concentration
Two running buffers phosphate and borate buffers at different pHs, were tested for the best resolution. In borate buffer, better resolved peaks with low background current were achieved.
Borate buffer concentration was changed from 10 to 40 mM. The EOF is reduced by increasing the buffer concentration, and vice versa. Moreover, Joule heating will occur upon using a high concentration of the buffer. 20 mM concentration of borate buffer was chosen as the optimum BGE to keep better peak shape within a reasonable migration time (Fig. S2).
4.6 SDS concentration
The concentration of SDS was investigated at concentrations ranging from 30 to 80 mM. The increase of SDS concentration from 50 to 80 mM, resulted in an improvement of separation pattern between analytes. At the same time, using 80 mM SDS leads to a significant increase in the current and the migration time. While a co-elution of the first eluting peaks was found at 10 mM SDS with a significant broadening peak. Therefore, 50 mM SDS was found to be the best concentration, providing moderate generated currents and reasonable analysis times (Fig. S3).
4.7 Applied voltage
The applied voltage affects the pseudo effective electrophoretic mobility, the electroosmotic mobility, the efficiency and hence the resolution of the analytes [58]. Under the optimum BGE conditions selected above, the influence of voltage (20–30 kV) on the migration time was studied. The EOF is increased by increasing the applied voltage, resulting in shorter analysis times and higher efficiency. Voltage of 25 kV was chosen as the optimal, as it maintains the separation within a reasonable time (Fig. S4).
4.8 Injection volume
The optimum injection volume was studied in order to gain highest sensitivity and improve the signal to-noise ratio [59]. For selection of the optimum injection parameters, samples were injected hydrodynamically at 50 mbar while injection times ranged from 5 to 20 s. After 10 s, the peak shapes were deformed and the resolution between peaks also decreased, thus 50 mbar for 10 s were the optimum injection parameters and corresponded to the highest sensitivity without compromising separation efficiency (Fig. S5).
4.9 Capillary cartridge temperature
Only a narrow range of temperatures can be examined [60]. Temperature effects were studied at 20, 25, and 30 °C. 25 °C was chosen because it produced the best resolution, peak shape, reasonable migration time and the generated current was not so high, about 35 μA. Increasing the temperature causing overlapped analytes peaks in addition to slight deformation.
4.10 Sample solvent
Different sample matrices were used with the same BGE composition (50 mM SDS and 20 mM disodium tetraborate buffer, pH 9.0). The matrices: 10 mM phosphoric acid (pH 2.15), borate buffer and water were used as the three sample matrices. The most favorable results were obtained upon dilution of stock solutions with water (Fig. S6).
4.11 Selection of the Internal Standard (I.S.)
Internal standard can enhance the method's precision significantly [61]. Atorvastatin, ezetimibe, saxagliptin and cromolyn sodium were tested. Cromolyn sodium was chosen as the I.S. because it provided higher resolution and better peak shape. To reduce major integration errors, it is preferred to employ a high concentration of the I.S. with a minimum signal-to-noise ratio of 30 [60]. The concentration of cromolyn sodium was kept constant at 1.0 μg mL−1 throughout the developed method.
5 Method validation
The designed MEKC method was validated in accordance with ICH guidelines [62].
5.1 Linearity and range
Each concentration was analyzed for three times and average peak area were calculated. Plotting the corrected peak area ratio (peak area of the drug/peak area of cromolyn sodium) alongside the drug concentration in μg mL−1 yielded a linear relationship. Table 1 summarizes the results of the statistical analysis [63] of the data and shows the linear range for each analyte. The correlation coefficients (r) are high enough (0.9998–0.9999) indicating the linearity of the calibration curves. Limits of quantitation and limits of detection (LOQ and LOD) were calculated mathematically according to ICH guidelines [62] and abridged in Table 1. The minimum concentration at which each drug could be detected was established practically by calculating signal to noise ratio 3:1. Similarly, the quantitation limit was obtained for each of the three drugs by calculating signal to noise ratio 10:1. The mathematical equations are represented as follows: LOD = 3.3 Sa/b, LOQ = 10 Sa/b, Where, Sa is the standard deviation of the intercept of a regression line and b is the slope.
Analytical performance data for the determination of the studied drugs by the proposed MEKC method
| Parameter | TNZ (at 315nm) | ORZ (at 315nm) | MNZ (at 315nm) | CIP (at 315nm) | DXM (at 230nm) | SPI (at 230 nm) |
| Linearity range (µg mL−1) | 0.5–5.0 | 0.5–5.0 | 1.0–10.0 | 1.0–10.0 | 1.0–10.0 | 1.0–10.0 |
| Intercept ± S.D. (Sa) | 0.33 ± 0.02 | −0.04 ± 0.03 | −6.7x10−3 ± 0.04 | 3x10−4 ± 0.018 | 0.0174 ± 0.02 | 0.0153 ± 2.9x10−2 |
| Slope ± S.D. (Sb) | 8.3 ± 0.74 × 10−3 | 0.0102 ± 0.96 × 10−2 | 7.6 ± 0.69 × 10−3 | 4.5 ± 0.29 × 10−3 | 5.9 ± 3.3 × 10−4 | 8.2 ± 0.44 × 10−3 |
| Correlation coefficient (r) | 0.9998 | 0.9998 | 0.9998 | 0.9999 | 0.9999 | 0.9999 |
| S.D. of residuals (Sy/x) | 0.03 | 0.038 | 0.05 | 2.29 × 10−2 | 0.026 | 0.03 |
| LOD (µg mL−1) | 0.09 | 0.095 | 0.20 | 0.13 | 0.12 | 0.12 |
| LOQ (µg mL−1) | 0.27 | 0.3 | 0.60 | 0.40 | 0.37 | 0.35 |
5.2 Accuracy
The data of analysis of the six cited drugs in their raw material were compared to those obtained by the previously reported methods [37, 64–67] to demonstrate the proposed method's accuracy. The data represented non-significant difference using Students' t-test and the variance ratio F-test [63] as shown in Tables 2–4.
Determination of TNZ and MNZ in raw material by the proposed MECK and reported methods, [66, 67]
| Ranges | Proposed method | Reported method, [66, 67] | ||||
| Taken (µg mL−1) | Found* (µg mL−1) | % Recovery | Taken (µg mL−1) | Found (µg mL−1) | % Recovery | |
| TNZ | 0.5 | 0.49 | 97.48 | 2.0 | 1.95 | 97.56 |
| 1.0 | 1.02 | 101.85 | 5.0 | 5.08 | 101.52 | |
| 2.0 | 2.02 | 101.01 | 10.0 | 9.97 | 99.67 | |
| 3.0 | 2.99 | 99.52 | ||||
| 4.0 | 3.95 | 98.78 | ||||
| 5 | 5.04 | 100.75 | ||||
| Mean % ± S.D. | 99.90 ± 1.62 | 99.58 ± 1.98 | ||||
| t-test | 0.26 (2.36) | |||||
| F-test | 1.51 (5.79) | |||||
| MNZ | 1.0 | 1.02 | 101.84 | 10.0 | 9.65 | 96.45 |
| 3.0 | 2.92 | 97.32 | 100.0 | 100.07 | 100.67 | |
| 5.0 | 5.06 | 101.13 | 200.0 | 199.68 | 99.84 | |
| 7.0 | 7.07 | 101.06 | ||||
| 8.0 | 7.95 | 99.34 | ||||
| 10.0 | 9.99 | 99.85 | ||||
| Mean % ± S.D. | 99.89 ± 0.92 | 98.99 ± 2.24 | ||||
| t-test | 0.85 (2.36) | |||||
| F-test | 1.87 (5.79) | |||||
*Average of three separate determinations.
The values between parentheses are the tabulated t and F values at P = 0.05 [63].
Determination of ORZ and CIP in raw material by the proposed MEKC and reported methods [64, 65]
| Ranges | Proposed method | Reported method [64, 65] | ||||
| Taken (µg mL−1) | Found (µg mL−1) | % Recovery | Taken (µg mL−1) | Found (µg mL−1) | % Recovery | |
| ORZ | 0.5 | 0.51 | 102.02 | 3.0 | 2.95 | 98.50 |
| 1.0 | 1.02 | 101.86 | 10.0 | 10.07 | 100.66 | |
| 2.0 | 1.99 | 99.71 | 18.0 | 17.94 | 99.69 | |
| 3.0 | 2.97 | 99.0 | ||||
| 4.0 | 3.96 | 98.98 | ||||
| 5.0 | 5.05 | 100.98 | ||||
| Mean % ± S.D. | 100.43 ± 1.38 | 99.62 ± 1.08 | ||||
| t-test | 0.88 (2.36) | |||||
| F-test | 1.63 (19.296) | |||||
| CIP | 1.0 | 0.99 | 99.98 | 10.0 | 9.81 | 98.08 |
| 2.0 | 1.97 | 98.46 | 30.0 | 30.38 | 101.28 | |
| 4.0 | 3.99 | 99.59 | 50.0 | 49.81 | 99.62 | |
| 6.0 | 6.09 | 101.44 | ||||
| 8.0 | 8.00 | 100.04 | ||||
| 10.0 | 9.96 | 99.60 | ||||
| Mean % ± S.D. | 99.85 ± 0.96 | 99.66 ± 1.60 | ||||
| t-test | 0.23 (2.36) | |||||
| F-test | 2.76 (5.79) | |||||
Determination of DXM and SPI at 230 nm in raw material by the proposed MEKC and reported methods [37, 64]
| Ranges | Proposed method | Reported method [37, 64] | ||||
| Taken (µg mL−1) | Found (µg mL−1) | % Recovery | Taken (µg mL−1) | Found (µg mL−1) | % Recovery | |
| DXM | 1.0 | 0.98 | 98.05 | 5.0 | 5.14 | 102.85 |
| 3.0 | 2.99 | 99.97 | 10.0 | 9.79 | 97.88 | |
| 5.0 | 5.02 | 100.35 | 20.0 | 20.07 | 100.37 | |
| 7.0 | 7.05 | 100.75 | ||||
| 9.0 | 8.94 | 99.30 | ||||
| 10.0 | 10.01 | 100.13 | ||||
| Mean %± S.D. | 99.76 ± 0.96 | 100.37 ± 2.49 | ||||
| t-test | 0.55 (2.36) | |||||
| F-test | 6.65 (5.79) | |||||
| SPI | 1.0 | 0.99 | 99.18 | 1.0 | 1.03 | 102.73 |
| 3.0 | 2.96 | 98.80 | 10.0 | 9.95 | 99.49 | |
| 5.0 | 5.03 | 100.67 | 20.0 | 20.03 | 100.13 | |
| 7.0 | 7.05 | 100.78 | ||||
| 9.0 | 8.99 | 99.89 | ||||
| 10 | 9.96 | 99.64 | ||||
| Mean %± S.D. | 99.83 ± 0.79 | 100.78 ± 1.72 | ||||
| t-test | 1.19 (2.36) | |||||
| F-test | 4.7 (5.79) | |||||
The values between parentheses are the tabulated t and F values at P = 0.05 [63].
5.3 Precision
Interday and Intraday precision of the proposed MEKC method were assessed and %RSD ranged from 0.37 to 2.07% (Table S1). These data were obtained by analysis of three concentrations within the linear range in the same day and in three successive days respectively.
5.4 Selectivity
The method's specificity was determined by examining any interference from the formulation's ingredients mentioned in the information leaflet. There was no interference from any additives, indicating that the suggested method is sufficiently selective.
5.5 Applications
5.5.1 Pharmaceutical formulations
By determining the investigated analytes in different co-formulated and single component formulations, the applicability of the new developed method was approved (Figs 3 and 4). As shown in Tables 5 & S2, the % recoveries for the studied drugs were in the range of 97.63%–102.56%. Moreover, the statistical tests revealed that the results obtained were in good agreement with those obtained using the comparison methods [37, 64–67].
Electropherograms for the analysis of: (A) Protozole 500 mg tablets; (B) Flagyl ® 500 mg tablets; (C) Ornidaz®500 mg tablets; (D) Ciprobay® 500 mg tablets; (E) Dexamethasone-MUP ampoules; (F) Spirex tablets
Citation: Acta Chromatographica 35, 3; 10.1556/1326.2022.01057
Electropherograms for the analysis of: (A) Tinifloxacin tablets; (B) Ciprodiazole tablets; (C) Laboratory prepared Co-formulated tablets Ciplox-OZ; (D) Zonacip otic drops
Citation: Acta Chromatographica 35, 3; 10.1556/1326.2022.01057
Assay results for the determination of the studied drugs in their co-formulated tablets by the developed MEKC method and the comparison methods [37, 64–67]
| Dosage form | Proposed method | Reference methods [37, 64–67] | |||||||
| Amount taken (µg mL−1) | % Founda | Amount taken (µg mL−1) | % Founda | ||||||
| Tinifloxacin® 500 mg tablets (500 mg CIP + 600 mg TNZ per tablet) | CIP | TNZ | CIP | TNZ | CIP | TNZ | CIP | TNZ | |
| 1.0 | 1.2 | 101.13 | 97.77 | 10.0 | 2.0 | 102.88 | 101.90 | ||
| 2.0 | 2.4 | 99.15 | 101.68 | 30.0 | 5.0 | 98.08 | 98.80 | ||
| 4.0 | 4.8 | 100.14 | 99.72 | 50.0 | 10.0 | 100.58 | 100.25 | ||
| Mean % | 100.14 | 99.72 | 100.51 | 100.32 | |||||
| ± S.D. | 0.99 | 1.96 | 2.40 | 1.55 | |||||
| t-testb | 0.25 (2.776) | 0.41 (2.776) | |||||||
| F-testb | 5.88 (19) | 1.59 (19) | |||||||
| Ciprodiazole® tablets (500 mg CIP + 500 mg MNZ per tablet) | CIP | MNZ | CIP | MNZ | CIP | MNZ | CIP | MNZ | |
| 2.0 | 2.0 | 98.97 | 102.13 | 10.0 | 10.0 | 102.88 | 97.65 | ||
| 5.0 | 5.0 | 100.66 | 98.63 | 30.0 | 100.0 | 98.08 | 100.45 | ||
| 10.0 | 10.0 | 99.88 | 100.25 | 50.0 | 200.0 | 100.58 | 99.89 | ||
| Mean % | 99.84 | 100.34 | 100.51 | 99.33 | |||||
| ± S.D. | 0.85 | 1.75 | 2.40 | 1.48 | |||||
| t-testb | 0.46 (2.776) | 0.76 (2.776) | |||||||
| F-testb | 8.06 (19) | 1.39 (19) | |||||||
| Laboratory prepared tablets CIP, ORZ [55] | CIP | ORZ | CIP | ORZ | CIP | ORZ | CIP | ORZ | |
| 1.0 | 1.0 | 98.33 | 101.97 | 10.0 | 3.0 | 102.88 | 96.18 | ||
| 3.0 | 3.0 | 101.11 | 98.68 | 30.0 | 10.0 | 98.08 | 102.11 | ||
| 5.0 | 5.0 | 99.67 | 100.39 | 50.0 | 18.0 | 100.58 | 99.43 | ||
| Mean % | 99.70 | 100.35 | 100.51 | 99.24 | |||||
| ± S.D. | 1.39 | 1.65 | 2.40 | 2.97 | |||||
| t-testb | 0.51 (2.776) | 0.56 (2.776) | |||||||
| F-testb | 2.98 (19) | 3.26 (19) | |||||||
| Zonacip otic drops each 1 mL contains (3.0 mg CIP and 1.0 mg DXM). | CIP | DXM | CIP | DXM | CIP | DXM | CIP | DXM | |
| 3.0 | 1.0 | 98.12 | 99.06 | 10.0 | 5.0 | 102.88 | 101.90 | ||
| 6.0 | 3.0 | 101.86 | 100.94 | 30.0 | 10.0 | 98.08 | 98.57 | ||
| 9.0 | 5.0 | 99.37 | 99.69 | 50.0 | 20.0 | 100.58 | 100.24 | ||
| Mean % | 99.78 | 99.90 | 100.51 | 100.24 | |||||
| ± S.D. | 1.90 | 0.96 | 2.40 | 1.67 | |||||
| t-testb | 0.41 (2.776) | 0.31 (2.776) | |||||||
| F-testb | 1.59 (19) | 3.03 (19) | |||||||
| Spirazole forte® tablets (379mgSPI+250 mg MNZ | SPI | MEZ | SPI | MNZ | SPI | MNZ | SPI | MNZ | |
| 1.52 | 1.0 | 98.36 | 101.04 | 1.0 | 10.0 | 97.44 | 97.65 | ||
| 4.56 | 3.0 | 100.91 | 99.43 | 10.0 | 100.0 | 100.48 | 100.45 | ||
| 9.12 | 6.0 | 99.82 | 100.12 | 20.0 | 200.0 | 99.88 | 99.89 | ||
| Mean % | 99.70 | 100.20 | 99.33 | ||||||
| ± S.D. | 1.28 | 0.81 | 1.48 | ||||||
| t-testb | 0.36 (2.776) | 1.12 (2.776) | |||||||
| F-testb | 1.58 (19) | 3.04 (19) | |||||||
aeach result is average of three readings. bthe figures between parentheses are the tabulated t and F values at P = 0.05.
6 Assessment of the greenness using GAPI tool
The assumptions about green chemistry's major purpose is to make both the environment and the operator safer. In today's world, determining a method's greenness and environmental impact is a vital stage in any established analytical method [68]. Green Analytical Procedure Index (GAPI) was used to investigate the suggested method's eco-friendly properties. To assess the greenness, it is necessary to cover the whole procedure items in 15 parameters [54]. Fifteen colored zones are represented by the GAPI pictogram (Fig. 5). In the proposed method; we don't use any organic solvent in the BGE to improve greenness. The procedure was also carried out without any previous derivatization, heating, or the inclusion of any other chemicals. Further dilutions were made with water. It might be viewed as a sensitive, environmentally friendly alternative to the previously reported methods.
GAPI assessment tool for the proposed MEKC method
Citation: Acta Chromatographica 35, 3; 10.1556/1326.2022.01057
7 Comparison with previously published methods
In Table 5, we compared the analysis of different tablet dosage forms using previous and current approaches. The statistical evaluation indicated no significant difference regarding accuracy and precision. However, when comparing the previous published methods [37, 64–67] with the proposed MEKC method, the current approach displays some advantages. Firstly, it is the first method for the concurrent determination of the six drugs. Moreover, the HPLC methods include the usage of organic modifiers as acetonitrile [37, 65, 66] or methanol [67]. Accordingly, the proposed method is a green alternative using lower sample volumes, micellar system and low time of analysis.
8 Conclusion
Simultaneous estimation of the drugs plays a very important role in pharmaceutical world and quality control. Therefore, a green, novel, precise and accurate MEKC method was investigated for the simultaneous estimation of TNZ, ORZ, MNZ, CIP, DXM and SPI in pharmaceutical dosage forms. The proposed approach is considered a green alternative due to the low sample volume, low solvent consumption and reduced analysis time, which accordingly decreases the generated waste. GAPI tool was used to assess the greenness of the method. In comparison to some previously published reports for the analytes, the suggested method is repeatable, fast, cost effective in addition to being direct sample injection without pretreatment.
Conflict of interest
The authors declared they have no conflict of interest.
Supplementary materials
Supplementary data to this article can be found online at https://doi.org/10.1556/1326.2022.01057.
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