Validation of RP-HPLC Method and UV Spectrophotometric Method for the Quantitative Determination of Aztreonam in Bulk and Pharmaceutical Dosage Form

INTRODUCTION

Aztreonam, a beta lactam antibiotic was introduced in 1978, isolated from a strain of Chromobacterium violaceum, is used to treat infections against Gram negative bacteria.¹ The Gram negative bacteria like E coli, P aeruginosa and P mirabillis are generally susceptible to this monobactam antibiotic.² It is inactive against Gram positive and anaerobic bacteria. Chemically aztreonam is (2S,3S)-3-{[(2Z)- 2-(2-Ammonio-1,3-thiazol-4-yl)-2-{[(2-carboxy-2- propanyl) oxy] imino} acetyl] amino}-2-methyl-4- oxo-1-azetidinesulfonate. The sulfonic acid moiety attached to the nitrogen on the beta lactam ring is responsible for activity against transpeptidase enzymes. The presence of amino thiazole side chain contributes to the activity against gram negative bacteria. The presence of alpha -methyl group stabilizes aztreonam against beta lactamase enzymes. It acts by blocking peptidoglycan crosslinking, thereby inhibiting the synthesis of the bacterial cell wall.³ It is marked as the most effective and safe medicines in health system by WHO list of essential medicines.⁴ Aztreonam is used for the treatment of bacteremia, pneumonia with cystic fibrosis, bone infection, intraabdominal infection, kidney infections, and urinary tract infection. The drug has poor oral bioavailability, so generally administered as an intravenous or intramuscular injection or inhaled using an ultrasonic nebulizer for the treatment of cystic fibrosis.

Numerous methods are reported for the analysis and determination of aztreonam in bulk and pharmaceutical dosage form.^5-7 The official monograph of USP39 mention the use of methanol and phosphate buffer in the ration of 1:4 of pH 3 as mobile phase in UV detector at 254 nm. This study aims at developing a simple, easy, rapid, accurate and specific UV Spectrophotometric and RP-HPLC method for qualitative and quantitative estimation of aztreonam in various formulations at pH 7.4 and pH 5.5 respectively.

MATERIALS AND METHODS

Aztreonam was gifted by FUAN pharmaceutical group Chongoing Bosen Pharmaceutical Co. Ltd., China. Methanol and water (HPLC-grade) were obtained from Himedia labs, Bangalore.

Membrane filter (0.22 µm) were obtained from Sigma–Aldrich. Formulation was procured from the local market having strength of 500 mg/ vial. Potassium dihydrogen phosphate, sodium hydroxide and rest of the chemicals were of analytical grade. All the measurements were made using HPLC grade water.

Chromatographic condition

Instrument used: Phenomenex (USA)

Temperature: Ambient

Column: C-8 Phenomenex HPLC column (Dimension 250X4.6 mm, 5µg)

Mobile Phase: Methanol: Phosphate Buffer pH 5.4(70:30)

Flow rate: 1 ml per min

Wavelength: 300 nm

Injection Volume: 20 µl

High performance liquid chromatography (HPLC) method was used to develop the standard graph of Aztreonam at different concentration of Aztreonam (2 -10 µg/ml) in the mobile phase. The instrument used was a Phenomenex (USA), consisting of a model LC AT VP intelligent solvent delivery pump, 7125-Rheodyne injector, a computerized system controller (with the Baseline chrome Tech N2000 software), and a UV SPD10A detector set at 300 nm. Chromatographic separation was performed using a C-8 Phenomenex HPLC column (Dimension 250X4.6 mm, 5 µg). The mobile phase consisted of Methanol: Phosphate buffer (70:30). The apparent pH of the mixed solvent system was adjusted to 5.4 ± 0.1.

The UV spectrophotometric method was performed in UV-Visible Spectrophotometer Tech comp 23.1.

Stock and working solution preparation

For HPLC method

Stock solution of Aztreonam at a concentration of 100 µg/ml was prepared in the mobile phase. From the stock several dilutions between 2-10 µg/ ml were prepared and injected into the column at a rate of 1 ml/min at injection volume of 20 µl. The calibration curve was plotted using area of retention time versus concentrations of aztreonam in µg/ml.

For UV method

A stock solution of Aztreonam in phosphate buffer pH 7.4 was prepared at a concentration of 100 µg/ml. It was further diluted with 100ml buffer to obtain a solution of concentration corresponding to 10 µg/ml. The absorbance of the resulting solution was scanned in the UV spectrometer in the range 200- 400 nm to determine the maximum wavelength (λ max).

Calibration curve was drawn at the λ max by varying the concentration of the diluted stock solution from 2-10 µg/ml. A calibration graph was generated with the absorbance and concentrations. All readings were taken in triplicate.

Method validation

The methods were validated as per the ICH guidelines on analytical process validation. The accuracy, precision, specificity, and robustness of the method were validated as per ICH guidelines Q(2) for validation of analytical procedure.⁸ The system suitability was carried for both the methods.

Determination of linearity

The calibration curve was obtained with five concentrations of the standard solution (2-10 µg/ ml for HPLC method and 2-10 µg/ml for UV spectrophotometric method. The linearity was evaluated by linear regression analysis using least square regression method.

Determination of precision and accuracy

The assay precision was carried out by withinday and inter-day study. Repeatability or within day precision was checked by assaying samples at same concentration and during the same day. The intermediate precision was studied by comparing the assays on three different days. For HPLC method interday and within day repeatability were evaluated for three solutions (0.5, 1,2 µg/ml) of different concentrations and were analyzed at different time points. For UV method six samples of aztreonam (1 to 6 µg/ml) in phosphate buffer pH 7.4 were analyzed at three different time points at 293nm. The % RSD was calculated for both the methods.⁹

Determination of specificity

A marketed product of Aztreonam, Azenam injection 500 mg/10 ml from Aristo Pharmaceuticals Pvt Ltd, India was taken, and specificity was determined in triplicate at a concentration of 2 µg/ ml in triplicate for HPLC method and 5µg/ml in triplicate for UV method, verified with the labelled content.

The average content and % RSD were calculated for each solution.

Determination of robustness

The robustness of the RP HPLC method was checked by allowing small deliberate change in injection flow rate (± 0.2 units) and wavelength (±1 unit).

The robustness of the spectrophotometric method was checked by allowing small change in pH of the solutions (± 0.2 units) and wavelength (± 1 unit).

% RSD was found out for both the method.

Limit of detection and limit of quantification

The limit of detection (LOD) and limit of quantification (LOQ) were determined according to ICH guidelines for both the methods.¹⁰

LOD= 3.3 * SE/ A (1)

LOQ= 10 * SE/ A (2)

SE=Standard error of Y intercept. A= Slope of the calibration curve

RESULTS AND DISCUSSION

For HPLC method

The mobile phase selection was based on peak parameters (symmetry, tailing), run time, ease of preparation and cost. The calibration curve showed a linearity in the range 2-10 µg/ml with a correlation coefficient of 0.9999, which is highly significant for the method as shown in Figure-1.

The LOD and LOQ were found to be 0.16 and 0.5 µg/ml, respectively, indicating high method sensitivity as expressed in table 1. The method precision was determined by withinday and interday and was expressed as % RSD. The result obtained showed RSD of 0.18% indicating good within day precision. Interday variability for three different days showed RSD of 0.83%. The accuracy of the method was determined, and the mean recovery which indicates the conformity between the true and found value, was found to be 100.03 ± 0.18% within day and 100.12 ± 0.84% inter-day as shown in table 2. The HPLC method was found to be specific to evaluate aztreonam in marketed formulations Azenam 500 mg injection from Aristro Pharmaceuticals Pvt. Ltd, India. The content was found at an average of 99.66 ± 1.28 % with a percetage RSD of 1.29. No interfering peaks of the excipients were observed in the chromatograms. Figure-2 reveals a typical chromatogram which was obtained from the analysis of a standard (8 µg/ml) and a sample solution of aztreonam from a vial (8 µg/ml) using the proposed method. The retention time observed was 1.95 m. This proved the ability of the developed method for rapid determination of the drug as a part of routine analyses. The method was found to be robust when the injection flow rate and wavelength were varied slightly. A slight change in injection flow rate and wavelength the %RSD was found to be less than 2% in both the cases as shown in table 3.

For UV method

The proposed spectrophotometric method in phosphate buffer pH 7.4 established a rapid and simple estimation of aztreonam. The solution of aztreonam at a concentration of 10µg/ml showed λmax was 293 nm as shown in figure 3. The calibration curves were constructed in the range of expected concentrations (2-10 µg/ml) as revealed in figure 4. The linear equation was found to be y=0.0085x + 0.015, with a correlation coefficient of 0.9988 (Table 1). LOD and LOQ were found to be 0.6 and 1.8 µg/ml. respectively, showing that the experimental values obtained for the determination of atenolol in the samples indicated a satisfactory intra-day variability (RSD of 0.78%) and interday variability (RSD of 1.12%). A good accuracy of the method was verified with a mean recovery of 99.98 ± 1.2% within day and 100.13 ± 3.21 % interday (Table 2). Finally, the method showed to be specific for the determination of aztreonam in marketed vials. The method was found to be robust when the pH of the solution and the wavelength varied slightly. A % RSD for change in both the process parameter showed less than 2 % as shown in table 3.

Comparison between HPLC and UV spectrophotometric method

Statistical analysis was done to compare both the analytical methods. ANOVA was applied and revealed no significant difference between the experimental values obtained in the analysis of aztreonam by the two methods. The calculated F-value (Fcalc = 0.082) was found to be less than the tabled F-value (Ftab= 3.19) at 1% significance level.

CONCLUSION

From the above study, it can be concluded that both the methods were found to be fast, reliable, precise, accurate and sensitive to analyze aztreonam in bulk and commercial dosage form. Therefore, the proposed methods can be used to do routine analysis of aztreonam in pharmaceuticals.