Abstract
As per the World Health Organization, 10% of medicines in low- and middle-income nations are of poor quality and pose a huge public health risk. The development and implementation of cost-effective, efficient and quick analytical methods to control the quality of these medicines is one of the immediate strategies to avoid such a situation. Hence, the main goal of this study was to develop and validate a simple, specific and precise new RP–HPLC method for simultaneous analysis of amoxicillin, ampicillin and cloxacillin in pharmaceutical formulations. The chromatographic analysis was achieved using Shodex C18 (250 × 4.6 mm, 5 μm) column with UV detection at 225 nm. The mobile phase was a gradient mixture of 30 mM phosphate buffer, pH 4.0 (mobile phase A) and acetonitrile (mobile phase B). Efficient separation of the three drugs was obtained using the final optimized chromatographic conditions. The developed method was validated for its specificity, linearity, precision, accuracy and robustness as per the ICH guidelines. The validation results showed that the method was specific, linear, precise, accurate and robust for the simultaneous determination of the three drugs. The developed method was applied to determine the content of the three drugs in pharmaceutical formulations. The assay results of the preparations showed that their drug content was within the pharmacopeial limit stipulated for each drug product. It can be concluded that the proposed method is suitable for simultaneous determination of amoxicillin, ampicillin and cloxacillin in pharmaceutical formulations in industries and regulatory laboratories.
Introduction
The β-lactam antibiotics are a class of broad-spectrum antibiotics consisting of all antibiotic agents that contain a β-lactam ring in their molecular structures. These include penicillin derivatives, cephalosporins, monobactams, and carbapenems. The penicillin derivatives include, among others, amoxicillin (AMOX), ampicillin (AMP) and cloxacillin (CLOX). They are extensively prescribed for the treatment of potentially life-threatening infections including peritonitis, respiratory tract infections, endocarditis, meningitis, skin and soft tissue infections which involve a wide range of bacteria species [1–4].
Substandard and counterfeit drugs are becoming huge worldwide problems. Counterfeit drugs are a global concern and are estimated to account for about 10% of all pharmaceuticals on the market. The problem is even worse in developing countries. WHO estimate showed that about 25% of medicines in developing countries are counterfeit with a figure as high as 50% in some places [5, 6]. Studies reported that out of 163 counterfeited antibiotics detected in the world until 2009, 50% were beta-lactams, 12% quinolones, 11% macrolides, lincosamides and synergistines, 7% cyclins and 20% other antibiotics [7]. Counterfeiting and substandard preparations of pharmaceuticals have serious consequences for consumers, health care providers, drug manufacturers and governments [5–8]. Because of these existing problems associated with counterfeit and substandard drugs in the market and for cost and speed of analysis implications to control their quality, technologies that can correctly and promptly analyze counterfeit and substandard products are critically needed. Quick and simple analytical methods for controlling counterfeit drugs are of vital necessity.
Various methods have been reported for analysis of the studied drugs individually, any combination of the three drugs and/or together with others [9–16]. However, to the best of our knowledge, no HPLC method has been reported in literature for the simultaneous determination of the three commonly used essential antibiotics: AMOX, AMP and CLOX in bulk or pharmaceutical dosage form so far. This research was therefore aimed at developing a simple, precise, accurate and robust reversed phase HPLC method for simultaneous determination of the three drugs. Comparing to the individual analysis of these β-lactam antibiotics as per their respective official methods in pharmacopoeias, developing a single HPLC method to analyze the three antibiotics simultaneously has significant benefits for rapid post marketing quality surveillance, most importantly in resource limited nations. Moreover, it reduces the overall cost and time of analysis significantly. The chemical structure of the three drugs is given in Fig. 1.
Chemical structure of amoxicillin (I), ampicillin (II) and cloxacillin (III)
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01043
Materials and methods
Chemicals and materials
Potassium dihydrogen orthophosphate (KH2PO4) and HPLC grade acetonitrile (ACN) were purchased from LOBA Chemie laboratory reagents & fine chemicals PLC (Mumbai, India). Analytical grade phosphoric acid was obtained from HiMedia laboratories PLC (Mumbai, India) and bi-distilled water was purchased from JOURILABS PLC (Addis Ababa, Ethiopia). In addition, pH meter was purchased from ADWA PLC (Szeged, Hungary) and analytical balance was procured from Mettler Toledo PLC (Greifensee, Switzerland).
Samples and reference substances
AMOX trihydrate (86.4%), AMP trihydrate (86.4%) and CLOX sodium (86.4%) working standards from Reyoung Pharma Co Ltd (Shanghai, China) were provided as a gift from the Ethiopian Food, Medicines, Health Care Administration and Control Authority (EFMHACA) and Addis Pharmaceutical Factory (APF), Ethiopia. The various formulated capsules of the drugs were purchased from retail pharmacies in Mekelle, Ethiopia.
HPLC system
Analysis was done using Agilent 1260 series HPLC system obtained from Agilent technologies (Waldbronn, Germany). The system is equipped with G1312B infinity binary pump, G4225A infinity high performance degasser, G1367E infinity high performance autosampler, G1316C infinity thermostat column compartment and G4212B multi wavelength detector (MWD). Integration and analysis of the chromatographic peaks were carried out using the chemstation software version B.04.
Chromatographic conditions
Optimum method for the separation and quantification of the three antibiotics was developed using Shodex C18 (250 × 4.6 mm, 5 µm) column kept at 30 °C. The mobile phase consisted of 30 mM potassium dihydrogen phosphate buffer with pH adjusted to 4.0 using diluted ortho phosphoric acid and acetonitrile in gradient mixtures. The gradient program [time (min)/%B (ACN)] was set as 0/10, 3/10 to 10/72 to 12/72 and back to 14/10. The injection volume was 20 µL with a flow rate of 1 mL min−1. UV detection was done at 225 nm. Solutions were filtered through a 0.45 µm membrane filter and ultrasonically degassed prior to use.
Sample preparation
Twenty capsules were taken for each of the three drugs and their mean weight determined. The powder equivalent to 20 mg AMOX trihydrate, 30 mg of AMP trihydrate and 20 mg of CLOX sodium were weighed accurately in 100 mL volumetric flask. Then, about 70 mL of the mobile phase, thereafter the solvent, was added to dissolve the sample with intermittent shaking for 15 min and then diluted to volume with the solvent to obtain a solution having a concentration of 0.2 mg mL−1 AMOX trihydrate, 0.3 mg mL−1 AMP trihydrate and 0.2 mg mL−1 CLOX sodium. Next, the solution was filtered through 0.45 µm membrane prior to injection to the HPLC system.
Similarly, working standards of 10 mg AMOX trihydrate, 15 mg AMP trihydrate and 10 mg CLOX sodium were weighed accurately and dissolved in 50 mL volumetric flask with the same solvent to obtain equivalent concentration as their respective sample solutions. The solution was filtered through 0.45 µm membrane filter. Finally, 20 µL of the solution was injected and analyzed in replicate determinations. The peak areas obtained were used to calculate concentration of the drugs.
Method validation
The developed LC method was validated according to the ICH guidelines [17]. The validation parameters evaluated include specificity, linearity, precision, accuracy and robustness of the method.
Results and discussion
Method development
In the development of the method for simultaneous analysis of AMOX, AMP and CLOX, several parameters which could affect the chromatographic selectivity were evaluated and optimized. The detection wavelength, composition of the organic phase, pH the of mobile phase, concentration of the buffer solution, column temperature, injection volume and gradient elution program were studied to achieve optimum separation of the three drugs.
Selection and optimization of mobile phase
Primarily, mobile phase used for the proposed method was evaluated based on the monographs of individual analysis of the drugs in pharmacopeias [2, 3] and from reports in literature for the analysis of either single or any combination of the three drugs [9–16]. Among others, dihydrogen potassium phosphate buffer and acetonitrile were commonly used as mobile phase in the analysis of these drugs. Henceforth, a mixture of acetonitrile-phosphate buffer was selected at initial experiments and used in the subsequent development and optimization of the proposed method at different proportions of the mixture and different pH of the buffer.
To begin with, the methods reported for simultaneous analysis of AMP and CLOX [10, 15] and for simultaneous analysis of AMOX and CLOX [11] were examined for the separation of the three drugs. The pH of the buffer was kept at 5.0 as stated in the pharmacopeial methods in the individual analysis of the drugs. A mixture of ACN and 30 mM KH2PO4 at pH 5.0 with isocratic mode (20:80 v/v) using Shodex C18 (250 × 4.6 mm, 5 µm) column was attempted by injecting the individual drugs and their equivalent mixture sequentially. Two problems encountered on the chromatogram of the analyte peaks. First, the peak shapes of AMP and CLOX were not good as they were with tailing, broad and generally not symmetric. Second, the retention time for CLOX was too long (20 min). In an attempt to solve these problems, the composition of the mobile phase was varied up to 65: 35 v/v ratio of buffer to ACN respectively. Although the retention time of CLOX was reduced to less than 20 min, another problem experienced as the peaks of AMOX and AMP co-eluted at this higher proportion of ACN. Over laid chromatograms of the above conditions are shown in Fig. 2.
Overlaid chromatograms of mix standards of AMOX, AMP and CLOX using isocratic elution at buffer (pH, 5.0): ACN ratio of A, (80:20 v/v) and B (65:35 v/v)
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01043
Subsequently, the effect of pH on peak shape and retention time was investigated. Keeping the initial composition of the mobile phase, pH values lower than the initial value of 5.0 (4.0 and 4.5) were investigated to monitor its effect on retention time and peak shape. Although there was no significant change in retention time, the peak shape was better than the previous conditions and it was best at pH of 4.0. Like the first attempt at pH 5.0, different compositions of the mobile phase up to 65:35 v/v of buffer to ACN respectively were investigated. Although the retention time of CLOX decreased, its peak was not symmetric enough. Besides, the peaks of AMOX and AMP co-eluted at higher amount of ACN. After careful examinations of the effect of different pH values of the buffer on separation efficiency of the three drugs, pH 4.0 was found to be better and hence selected to be used throughout the development and optimization of the method. Maintaining the mobile phase pH at desirable value for these has paramount importance. The reason why the pH is so significant in HPLC analysis is because when the pH of the buffer is equal or set close to the pKa of the analyte, the analytes exist in solution in both ionized and unionized forms proportionally. As a result, analytes of the same molecule have different retention/affinity while passing through the column which will result in poor peak shape and inconsistent chromatography [18, 19].
Furthermore, the buffer concentration was investigated at concentrations of 10, 20 and 30 mM to examine its effect on retention time and peak shape of the analytes. The effect of the concentrations of the buffer at pH 4.0 on retention time of the drugs was not significant. Nevertheless, its effect on peak shape explained by peak symmetry and peak efficiency was slightly variable among the concentrations. Better separation was obtained at buffer concentration of 30 mM and as a result it was selected for further development and optimization of the method.
The flow rate was set at 1.0 mL min−1 which is the same as stipulated in each monograph in the pharmacopeias. Considering the effect on column back pressure at flow rate greater than 1 mL min−1 and its effect on increasing retention of the drugs at less than 1 mL min−1, the indicated flow rate was used throughout the study. The final optimized chromatogram with this flow rate was good in terms of peak shape and retention time while keeping the pump pressure effectively at less than 150 bar. The injection volume was also varied between 10
Selection of stationary phase
Like the selection of mobile phase, pharmacopeias and different literatures were reviewed to select stationary phase (column) for the proposed method. The most commonly reported columns for the analysis of the drugs individually, any combination of the three and/or with others were C18 (250 × 4.6 mm, 5 µm) columns. Hence, this column was selected and used throughout the study. However, C18 columns of 150 mm with the same internal diameter and particle size could also be possible options.
Selection of column temperature and detection wavelength
Different column temperatures (25–30 °C) were investigated to improve the peak symmetry of the three drugs. A column temperature of 30 °C showed better peak efficiency for the three drugs and hence it was used throughout the development and validation of the method. To determine a wavelength with better intensity for the three drugs, investigation of wavelength from 210 nm to 254 nm was done. A wavelength of 225 nm was found to be the most sensitive to the three drugs.
Gradient program and further optimization of the method
Preceding to the introduction of gradient elution for the developed method, separation of the three drugs was attempted with mobile phases of constant composition (isocratic elution). Though, isocratic separation works well for many samples and it is the easiest and most suitable form of liquid chromatography, no single mobile phase composition can provide a generally satisfactory separation for some samples [20] as it was in our case. The later peaks were wider and have unacceptably long retention times. The goal of gradient elution was therefore to reduce retention time, peak broadening, peak co-elution and peak tailing experienced during isocratic separation. Because of the increase in mobile phase strength during the time a peak is eluted in gradient elution, the tail of the peak moves faster than the peak front, with a resulting reduction in peak tailing and peak width.
The use of higher percentage of acetonitrile (35%) using isocratic mode partly addressed the problems, but at the same time it caused another problem as the peaks of AMOX and AMP co-eluted at higher amount of ACN. In addition, the peak of CLOX was not sharp, symmetric and its retention did not decrease significantly. Hence it was concluded that the isocratic mode cannot be used above 35% v/v of ACN. Consequently, the shift to gradient elution was necessary and the optimization of the method proceeded. Different gradient programs (Table 1) were attempted with the aim of reducing broadening, tailing and retention time of the later peaks. Considering the conditions in the isocratic separation attempt, a gradient program shown in Table 1 program I was used as a starting composition of the mobile phase to further optimize the method.
Different gradient programs used during development of the method
| Time (min) | % mobile phase A | % mobile phase B | Remark |
| Gradient program I (mobile phase A KH2PO4 buffer, mobile phase B ACN) | |||
| 0–2 | 87 | 13 | Isocratic |
| 2–5 | 87→70 | 13→30 | Linear gradient |
| 5–8 | 70 | 30 | Isocratic |
| 8–15 | 70→60 | 30→40 | Linear gradient |
| 15–18 | 60 | 40 | Isocratic |
| 18–25 | 87 | 13 | Re-equilibration |
| Gradient program II (mobile phase A KH2PO4 buffer, mobile phase B ACN) | |||
| 0–2 | 87 | 13 | Isocratic |
| 2–10 | 87→45 | 13→55 | Linear gradient |
| 10–15 | 45 | 5→5 | Isocratic |
| 15–18 | 45→87 | 55→13 | Linear gradient |
| 18–20 | 87 | 13 | Re-equilibration |
| Gradient program III (mobile phase A KH2PO4 buffer, mobile phase B ACN) | |||
| 0–2 | 87 | 13 | Isocratic |
| 2–10 | 87→35 | 13→65 | Linear gradient |
| 10–15 | 35 | 65 | Isocratic |
| 15–18 | 35→87 | 65→13 | Linear gradient |
| 18–20 | 87 | 13 | Re-equilibration |
The separation of the analytes using this gradient program resulted in a longer retention time for CLOX and the peak for AMP was not symmetric. The resulting chromatogram is shown in Fig. 3. To further optimize the separation efficiency, the strength of the mobile was increased by increasing the proportion of ACN as shown in program II, Table 1. Results of this gradient program (Fig. 3) showed a significant reduction in the retention time of CLOX but the AMP peak remained asymmetric. Furthermore, the total analysis time still needed to be reduced. Subsequently, another gradient program with stronger proportion of ACN as shown in program III in Table 1 was investigated in an attempt to get better peak shapes with shorter retention times. The resulting chromatograms for this gradient program are shown in Fig. 3. While the retention time and peak shape of CLOX was modified, another problem encountered as the peak of AMOX co-eluted with a small impurity peak which was eluting preceding it in the previous gradients.
Overlaid chromatograms of mix standards of AMOX, AMP and CLOX on a Shodex C18 (250 × 4.6 mm, 5 µm) column using gradient program I (I), gradient program II (II) and gradient program III (III)
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01043
Afterwards, various gradient programs were systematically examined to optimize gradient program III. The condition was optimized by decreasing the proportion of ACN in the initial isocratic run in gradient program III to 10% and increasing this run time to 3 min so that the preceding impurity peak and the AMOX peak have enough resolution. Finally, the gradient program shown in Table 2 was selected as the optimized mobile phase gradient mixture as it gives improved peak shape with reasonable retention times of the three drugs. Representative chromatogram of this optimized method obtained at 225 nm is shown in Fig. 4.
Optimized gradient program (mobile phase A KH2PO4 buffer pH = 4.0, mobile phase B ACN)
| Time (min) | % mobile phase A | % mobile phase B (ACN) | Remark |
| 0–3 | 90 | 10 | Isocratic |
| 3–10 | 90→28 | 10→72 | Linear gradient |
| 10–12 | 28 | 72 | Isocratic |
| 12–14 | 28→90 | 72→10 | Linear gradient |
| 14–16 | 90 | 10 | Re-equilibration |
Chromatogram of mixed standards of AMOX, AMP and CLOX on a Shodex C18 (250 × 4.6 mm, 5 µm) column, thermostatted at 30 °C, at detection wavelength of 225 nm using the optimized gradient elution program
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01043
Method validation
Once the proposed method was developed and optimized, its validation was conducted according to the ICH guidelines [17] for its specificity, linearity, precision, accuracy and robustness.
Specificity
The developed method was tested for its specificity to make sure that no other interferences from the solvent and matrix were present in the chromatograms of the three drugs at the specified wavelength. The blank (mobile phase and solvent), placebo (excipients in the capsule formulation of the three drugs), and the references of the three drugs were injected and their chromatograms compared. As it can be seen from Fig. 5, there was no interference due to the blank and/or excipients for the main peak of each of the drugs. Hence, it can be concluded that the developed method is specific for the simultaneous determination of the three drugs.
Overlaid chromatograms of A: blank, B: placebo and C: reference drugs on a Shodex C18 (250 × 4.6 mm, 5 µm) column, thermostatted at 30 °C, using the optimized gradient elution program
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01043
Linearity
The linearity of the response (peak area) of the drugs was determined at six concentration levels ranging from 25% to 150% of the assay concentration for each of the three drugs. The assay concentration (100%) was 0.2, 0.3 and 0.2 mg mL−1 for AMOX, AMP and CLOX respectively. The six concentration levels were in the range of 0.05–0.3 mg mL−1 for AMOX, 0.075–0.45 mg mL−1 for AMP and 0.05–0.3 mg mL−1 for CLOX. Each of the six concentrations was prepared by diluting standard stock solution of 0.4, 0.6, 0.4 mg mL−1 for AMOX trihydrate, AMP trihydrate, and CLOX sodium working standards respectively using the solvent. The coefficients of determination (R2) values were greater than 0.999 for the three drugs. This indicates that the detector response was linear in the above specified concentration ranges.
Precision
Precision was determined as % RSDs of the peak areas of the drugs. The % RSD for the repeatability, intra- and inter- day precisions were less than 2% for the respective drugs at the assay concentration of 0.2, 0.3 and 0.2 mg mL−1 for AMOX, AMP and CLOX respectively. Results of the % RSD values of repeatability and intermediate precision studies (Table 3) showed that the method is precise for simultaneous determination of the three drugs in bulk and pharmaceutical formulation.
Results of precision study
| Precision level | % RSD | ||
| AMOX | AMP | CLOX | |
| Repeatability (n = 6) | 0.4 | 0.3 | 0.1 |
| Intra–day precision (n = 6) | 0.7 | 1.1 | 0.7 |
| Inter–day precision (n = 18) | 1.2 | 1.1 | 1.0 |
Accuracy
Accuracy was evaluated as percent recovery of the added standards of the three drugs at the concentration levels of 80%, 100% and 120% of the assay concentration of each drug to the equivalent weights of the excipients (placebo) in their respective formulation. As shown in Table 4, the percentage recovery of the three drugs were in the range of 98–102% indicating the good accuracy of the optimized method for simultaneous determination of AMOX, AMP and CLOX in dosage forms.
Results of recovery study
| Conc. level (%) | % Recovery (n = 6, %RSD) | ||
| AMOX | AMP | CLOX | |
| 80 | 100.8 (0.6) | 100.7 (0.5) | 101.8 (1.2) |
| 100 | 99.2 (0.5) | 99.1 (0.6) | 100.01 (0.1) |
| 120 | 99.8 (0.8) | 98.2 (1.2) | 100.9 (0.6) |
Robustness
Robustness of the developed LC method was performed by means of experimental design analysis using Minitab software version 17. In this study, three chromatographic factors: pH of mobile phase, concentration of buffer (Buff), column temperature (Temp) and their interactions were examined. A two-level full factorial design was applied with a number of runs given by the formula 2k + n, where k is the number of factors and n is the number of center points [21–23]. Small but deliberate changes in column temperature, buffer concentration, and pH of mobile phase was made to the method. The levels of these selected factors were defined symmetrically around the nominal value (central value). The interval selected between the extreme levels represented the limits between which the factors are expected to vary when the method is applied to actual works in different setup and operating conditions. The selection of the levels was done based on practical considerations and taking the precision or uncertainty of the operating instruments into account [21]. The lower, nominal and higher values of each factor in the design is shown in Table 5. The P-value from full factorial analysis was used to determine the effect of the factors and their interactions on the response (peak area). A level of significance (P-value) of
Parameters and their values in robustness study
| Parameter | Low value (−) | Nominal value (0) | High value (+) |
| pH | 3.8 | 4 | 4.2 |
| Column Temp (ºC) | 28 | 30 | 32 |
| Weight of KH2PO4 in 1 L water (g) | 3.88 | 4.08 | 4.28 |
According to the formula, a total of 11 experiments with different combinations were conducted to investigate the effect of the selected parameters. The three factors selected were the most prominent chromatographic parameters that showed significant effect on peak shape and the overall separation efficiency. Peak area of the drugs was taken as response factor. As shown in Table 6, there was no significant change in the peak areas as a result of the variations made in the investigated parameters (P value
Analysis of variance for pH, Temp, weight of buff and their interactions on the peak area of amoxicillin, ampicillin and cloxacillin
| Source | ¥DF | *Adj SS | **Adj MS | F-value | P-value |
| Amoxicillin | |||||
| pH | 1 | 91.13 | 91.13 | 0.14 | 0.729 |
| Temp | 1 | 1,579.22 | 1,579.22 | 2.5 | 0.212 |
| Wt of Buff | 1 | 172.98 | 172.98 | 0.27 | 0.637 |
| pH*Temp | 1 | 400.44 | 400.4 | 0.63 | 0.484 |
| pH*Wt of Buff | 1 | 609.01 | 609.01 | 0.97 | 0.398 |
| Temp* Wt of Buff | 1 | 865.28 | 865.28 | 1.37 | 0.326 |
| pH*Temp* Wt of Buff | 1 | 361.8 | 361.8 | 0.57 | 0.504 |
| Ampicillin | |||||
| pH | 1 | 2,135.3 | 2,135.31 | 1.1 | 0.371 |
| Temp | 1 | 311.3 | 311.25 | 0.16 | 0.716 |
| Wt of Buff | 1 | 5,989.7 | 5,989.65 | 3.08 | 0.177 |
| pH*Temp | 1 | 0.1 | 0.06 | 0.00 | 0.996 |
| pH*Wt of Buff | 1 | 1,292.9 | 1,292.86 | 0.67 | 0.474 |
| Temp* Wt of Buff | 1 | 0.4 | 0.36 | 0.00 | 0.990 |
| pH*Temp* Wt of Buff | 1 | 1,252.5 | 1,252.5 | 0.64 | 0.481 |
| Cloxacillin | |||||
| pH | 1 | 798.0 | 798.0 | 0.31 | 0.616 |
| Temp | 1 | 12,920.3 | 12,920.3 | 5.05 | 0.110 |
| Wt of Buff | 1 | 11,272.5 | 11,272.5 | 4.41 | 0.127 |
| pH*Temp | 1 | 2,675.5 | 2,675.5 | 1.05 | 0.382 |
| pH*Wt of Buff | 1 | 2,453.5 | 2,453.5 | 0.96 | 0.400 |
| Temp* Wt of Buff | 1 | 33.2 | 33.2 | 0.01 | 0.916 |
| pH*Temp* Wt of Buff | 1 | 15.4 | 15.4 | 0.01 | 0.943 |
¥ degree of freedom, *Adjusted sums of squares, **Adjusted mean squares.
In order to better estimate the effect of the factors/predictors on the response, 3D response surface plots as shown in Fig. 6 were constructed using factorial design analysis on NCSS 12 statistical software. This response surface plot is a graph of the response function as a function of two of the three factors while the third factor is kept constant at its central value. The 3D surface plot helps to easily visualize the effects of two factors on the outcome variable. The plot contains the predictors on the x- and y-axes and a continuous surface that represents the response values on the z-axis which is the peak area of the drugs in our case. Two of the three selected chromatographic factors were varied while the third factor kept constant at its central value to see the effect of the predictors on the peak areas of the three drugs.
Response surface plots showing the influence of pH, temperature and concentration of buffer on peak areas of AMOX, AMP and CLOX by varying two of the three factors selected for robustness study while holding the third factor at its central value using factorial design analysis on NCSS 12 statistical software
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01043
The peak area of AMOX in the 3D response surface plot (Fig. 6) increases as any of two variables increases while the third variable kept at its central value until the central values with the highest value corresponding to the central values. After the central values, the area decreases with increment of the predictor variables. Similarly, the peak area of AMP showed in 3D response surface plot (Fig. 6) increases as a function of increment of two of the three predictor variables while the third variable is held constant as its central value. The highest value of AMP area corresponds to the central values of the predictor variables. After the central values, the area decreases with increment of the two variables. In contrary to the effect on peak areas of AMOX and AMP, the mobile phase pH has a negative effect on peak of CLOX. Divergently, increasing buffer concentration and column temperature have positive effect on peak area of CLOX. However, all the changes in peak areas as a function of the parameters was found to be insignificant (P-value>0.05) in Minitab ANOVA analysis shown in Table 6.
Some methods have been reported for analysis of the studied drugs with other penicillin antibiotics in different biological samples [24–26]. Nevertheless, to the best of our knowledge, no HPLC-UV method has been reported for the simultaneous determination of the three commonly used essential β-lactam antibiotics in pharmaceutical dosage forms so far. Moreover, these methods employed relatively more sophisticated techniques such as capillary HPLC [24] and HPLC-MS [25] compared to the proposed new method (HPLC-UV) where such techniques are rarely available in resource limited countries. Besides, the developed method has the advantage of being simple and fast compared to the method reported by L.K. Sørensen et al [26]. where HPLC-UV was employed to determine seven penicillins in muscle, liver and kidney tissues from cattle and pigs. Hence, the proposed method could be helpful for routine quality evaluation of AMOX, AMP and CLOX simultaneously in industries and regulatory laboratories.
Application of the method: assay of commercial samples
The developed method was applied for determination of capsule preparation of the drugs commercially available in the local markets of Mekelle, Ethiopia. In addition, assay on powder for injection formulations for AMP and CLOX with another capsule formulation for AMOX was conducted. The results are summarized in Table 7 as percentage content w/w of the drugs and representative chromatogram of the capsule formulations is shown in Fig. 7. The products tested were found within their pharmacopeial limits and hence confirmed as being compendial quality with regard to content of their active pharmaceutical ingredients.
Assay results of commercial samples
| Dosage form | Component | Claimed conc. (mg mL−1) | Conc. found (mg mL−1) | % content (w/w) |
| Capsule | AMOX | 0.2 | 0.196 | 98 |
| Capsule | AMP | 0.3 | 0.303 | 101 |
| Capsule | CLOX | 0.2 | 0.204 | 102 |
| Capsule | AMOX | 0.2 | 0.192 | 96 |
| Powder for injection | CLOX | 0.3 | 0.194 | 97 |
| Powder for Injection | CLOX | 0.2 | 0.206 | 103 |
Chromatogram of commercial samples (capsule) on a Shodex C18 (250 × 4.6 mm, 5 µm) column, thermostatted at 30 °C, using the optimized gradient elution program
Citation: Acta Chromatographica 35, 2; 10.1556/1326.2022.01043
Conclusion
The developed LC method has the advantages of being simple, specific, precise, accurate and rapid for simultaneous determination of AMOX, AMP and CLOX in bulk and pharmaceutical formulations. Moreover, the method has the benefit of being economical in terms of organic solvent consumption and time of analysis compared to the separate analysis of the three drugs. The developed method was validated for its specificity, linearity, precision, accuracy and robustness according to the ICH guidelines. The validation results showed that the method is specific, precise, accurate robust and linear in the specified ranges for the determination of AMOX, AMP and CLOX. The developed method was applied for the assay of commercial samples (capsule and powder for injection) and satisfactory results were obtained. The developed method is simple, economical and fast compared to the individual analysis of the drugs as per the official compendia. Henceforth, it can be concluded that this new method could be applied for quality evaluation of AMOX, AMP and CLOX simultaneously in industries and regulatory laboratories.
Data availability
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of interest
There authors declare no conflicts of interest regarding the publication of this manuscript.
Funding statement
This work was supported by Mekelle University.
Acknowledgments
The authors would like to thank Mekelle University for financial support to conduct this study. We also gratefully thank the Ethiopian Food and Drug Administration and Addis Pharmaceutical Factory for providing us the reference standards of the three drugs.
References
- 1.↑
Goodman, L.; Gilman, A.; Brunton, L.; Lazo, J.; Parker, K. Goodman & Gilman's the Pharmacological Basis of Therapeutics, 10th ed.; McGraw-Hill: New York, USA, 2006.
- 3.↑
United States Pharmacopeia. United States Pharmacopeial Convention. Rockville MD, Maryland, USA, 2010.
- 4.
Rolinson, G.; Eddes, A. The 50th anniversary of the discovery of 6-aminopenicillanic acid. Int. J. Antimicrob. Agents 2007, 29, 3–8.
- 5.↑
World health organization. Report of the Situation of Counterfeit Medicines Based on Data Collection Tool; WHO: Geneva, 2010.
- 6.↑
World health organization. The Safety of Medicines in Public Health Programmes; WHO: Geneva, 2018.
- 7.↑
Delepierre, A.; Gayot, A.; Carpentier, A. Update on counterfeit antibiotics worldwide. Public health risks, Medecine et maladies infectieuses 2012, 42, 247–255.
- 8.
Nuhu, A. Recent analytical approaches to counterfeit drug detection. J. Appl. Pharm. Sci. 2011, 01, 06–13.
- 9.↑
Ma, S.; Shi, Y.; Haixia, L.; Shang, X. Development and validation of a HPLC with solid phase clean-up and pre-column derivatization method for the simultaneous determination of three β- lactam antibiotics residues in eggs. World J. Pharm. Res. 2014, 3, 33–44.
- 10.↑
Shakoor, O.; Taylor, R. Analysis of ampicillin, cloxacillin and their related substances in capsules, syrups and suspensions by high performance liquid chromatography. Analyst 1996, 121, 1473–1477.
- 11.↑
Ramesh, K.; Guhle, A. Design, development and validation of amoxicillin and cloxacillin formulation by HPLC. Int. J. Pharmaceutics Drug Anal. 2016, 4, 448–451.
- 12.
Camara, M.; Gallego, A.; Garcinuno, R.; Fernandez, P.; Durand, J.; Sanchez, P. An HPLC-DAD method for the simultaneous determination of nine β-lactam antibiotics in ewe milk. Food Chem. 2013, 141, 829–834.
- 13.
Legrand, T.; Vodovar, D.; Tournier, N.; Khoudour, N.; Hulin, A. Simultaneous Determination of eight β-Lactam antibiotics: amoxicillin, cefazolin, cefepime, cefotaxime, ceftazidime, cloxacillin, oxacillin, and piperacillin in human plasma by using UHPLC with UV detection. Antimicrob. Agents Chemother. 2016, 60, 4734–4742.
- 14.
Khalid, A.; Rashood, A. Simultaneous determination of ampicillin and dicloxacillin in pharmaceutical formulations by HPLC. J. Liquid Chromatogr. 2006, 16, 2891–2897.
- 15.↑
Thushara, B.S. Method development and validation for simultaneous estimation of ampicillin and cloxacillin capsules by using RP-HPLC. World J. Pharm. Med. Res. 2017, 3, 160–163.
- 16.
Hameed, E.; Salam, R.; Hadad, G. Development of an optimized HPLC method for the simultaneous determination of six compounds containing β-lactam ring in human plasma and urine using experimental design methodology. R. Soc. Chem. Adv. 2016, 6, 9465–9474.
- 17.↑
ICH. ICH Guidelines Q2 (R1) Validation of Analytical procedures: Text and Methodology; EMA, Ed.; International Council for Harmonization: London, 2006.
- 18.↑
Heyrman, A.N.; Henry, A.R. Importance of Controlling Mobile Phase pH in Reversed Phase HPLC. Keystone Technical Bulletin 1999.
- 19.↑
Schoenmakers, P.J.; Molle, S.V.; Hayes, C.; Uunk, L. Effects of pH in reversed-phase liquid chromatography. Analytica Chim. Acta 2011, 250, 1–19.
- 20.↑
Snyder, L.; Dolan, J. High Performance Gradient Elution: the Practical Application of the Linear Solvent Strength Model, 2nd ed.; Wiley: New Jersy, 2007.
- 21.↑
Vander, H.Y.; Questier, F.; Massart, D.L. Ruggedness and robustness testing. J. Chromatogr. 2007, 1158, 138–157.
- 22.
Douglas, M. Design and Analysis of Experiments, 8th ed.; Wiley: USA, 2013.
- 23.
Gary, W.A First Course in Design and Analysis of Experiments; Library of congress cataloging, University of Minnesota (USA), 2000.
- 24.↑
Bailón-Pérez, M.I.; García-Campaña, A.M.; del Olmo-Iruela, M.; Gámiz-Gracia, L.; Cruces- Blanco, C. Trace determination of 10 β-lactam antibiotics in environmental and food samples by capillary liquid chromatography. J. Chromatogr. A 2009, 1216, 8355–8361.
- 25.↑
Straub, R.F.; Voyksner, R.D. Determination of penicillin G, ampicillin, amoxicillin, cloxacillin and cephapirin by high-performance liquid chromatography-electrospray mass spectrometry. J. Chromatogr. 1993, 647, 167–181.
- 26.↑
Sørensen, L.K.; Snor, L.K; Elkӕr, T.; Hansen, H. Simultaneous determination of seven penicillins in muscle, liver, and kidney tissues from cattle and pigs by a multiresidue high-performance liquid chromatographic method. J. Chromatogr. B 1999, 734, 307–318.
