Estimation of ampicillin trihydrate in bulk and formulation by first order derivative area under curve UV-Spectrophotometric methods

Simple, fast and reliable spectrophotometric methods were developed for determination of Ampicillin Trihydrate in bulk and pharmaceutical dosage forms. The solutions of standard and the sample were prepared in Methanol. The quantitative determination of the drug was carried out using the second order Derivative Area under Curve method values measured at 224-231nm. Calibration graphs constructed at their wavelengths of determination were linear in the concentration range of Ampicillin Trihydrate using 5-25μg/ml (r2=0.997) for first order Derivative Area under Curve spectrophotometric method. The proposed methods have been extensively validated as per ICH guidelines. There was no significant difference between the performance of the proposed methods regarding the mean values and standard deviations. The developed methods were successfully applied to estimate the amount of Ampicillin Trihydrate in pharmaceutical formulations.

To our notice, no UV-spectrophotometric method using First Order Derivative Area under Curve has been reported for the determination of Ampicillin Trihydrate in bulk and tablets. Hence an attempt has been made to develop new First Order Derivative Area under Curve spectrophotometric method for estimation of Ampicillin Trihydrate in bulk and pharmaceutical formulations with good accuracy simplicity, precision and economy.

Derivative Spectrophotometric Methods
The first derivative spectrophotometry was used in the wavelength ranges from 241 and 251 nm.
[dA/dλ= f(λ ): first order The first derivative spectrum of an absorption band is characterized by a maximum, a minimum, and a cross-over point at the λ max of the absorption band. [ Where, α is area of portion bounded by curve data and a straight line connecting the start and IJASR|VOL 01|ISSUE 05|2015 www.ssjournals.com end point, β is the area of portion bounded by a straight line connecting the start and end point on curve data and horizontal axis, λ1 and λ2 are wavelength range start and end point of curve region. [9]

Apparatus and instrumentation
A shimadzu 1800 UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for all spectral measurements. Single Pan Electronic balance (CONTECH, CA 223, India) was used for weighing purpose. Sonication of the solutions was carried out using an Ultrasonic Cleaning Bath (Spectra lab UCB 40, India). Calibrated volumetric glassware (Borosil®) was used for the validation study.

Materials
Reference standard of Ampicillin Trihydrate API was supplied as gift sample by Lupin Laboratory Park, Aurangabad. Methanol was obtained from Research-Lab Fine Chem Industries, Islampur, Mumbai, and Maharashtra. Capsule sample with label claim 500 mg per Capsule were purchased from local market Mangalwedha, Solapur, Maharashtra, India.

Method development 2.5.1 Preparation of Standard and Sample Solutions
Stock solution of 10μg/ml of Ampicillin Trihydrate was prepared in Methanol, for First Order Derivative Area under Curve spectrophotometric analysis. The standard solutions were prepared by dilution of the stock solution with Methanol in a concentration range of 5, 10, 15, 20 and 25μg/ml with Methanol for First Order Derivative Area under Curve spectrophotometric methods. Methanol was used as a blank solution.

Calibration curve for Ampicillin Trihydrate
The dilutions were made from Standard Stock solution to get concentration of 5, 10, 15, 20, and 25µg/ml respectively. These solutions were scanned from 400 to 200 nm and First Order Derivative Area under Curve values was integrated in the range of 224-231 nm. The calibration curve was plotted between areas under curve values against concentration.
www.ssjournals.com Then solution was further diluted up to the mark with Methanol. The solution was filtered using Whatmann filter paper no. 41, first 5 ml of filtrate was discarded. This solution was further diluted to obtain 10µg/mL solution with water, subjected for UV analysis using Methanol as blank. This procedure was repeated three times.

Method Validation
The above method was validated for various parameters such as Accuracy, Linearity, Precision, Limit of detection (LOD) and Limit of Quantitation (LOQ) according to ICH guideline.

Accuracy
The accuracy for the analytical method was evaluated at 80%, 100% and 120% levels of 25µg/ml Sample solution. First Order Derivative Area under curve (AUC) was measured in wavelength range 224-231 nm and results were obtained in terms of percent recovery. Three determinations at each level were performed and % RSD was calculated for each level.

Precision
The precision of an analytical procedure expresses the closeness of an agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions intraday precision was studied by integrating area of standard solution of 25µg/ml concentration at six independent series in the same day. Interday precision studies were performed by integrating area of standard solution of 25µg/ml concentration on three consequent days. The %RSD Was calculated.

Limit of Detection and Limit of Quantification
The Limit of Detection (LOD) is the smallest concentration of the analyte that gives the measurable response. LOD was calculated using the following formula LOD = 3.3 σ /S The Limit of Quantification (LOQ) is the smallest concentration of the analyte, which gives response that can be accurately quantified. LOQ was calculated using the following formula LOQ = 10 σ/S Where, σ is standard deviation of the response and S is the slope of the calibration curve. LOD& LOQ of Ampicillin Trihydrate was found to be 0.59µg/ml &1.47µg/ml respectively. www.ssjournals.com

Results and Discussion
The UV visible spectroscopic method for the Ampicillin Trihydrate by First order derivative Area under Curve was found to be simple, accurate, economical and reproducible. The drug concentrations were found to be linear in the range of 05-25 µg/ml and the correlation coefficient value of 0.997 indicates that developed method was linear. For Precision the percent relative standard deviation (% RSD) was found to be 0.0481 while, intra-day and inter-day precision results in terms of percent relative standard deviation values were found to be 0.7581 and 0.7958 respectively thus the method is observed as precise. The accuracy of the method was assessed by recovery studies at three different levels i.e. 80%, 100%, 120%. The values of standard deviation were satisfactory and the recovery studies were close to 100%. The % RSD value is ≤ 2 indicates the accuracy of the method. The Limit of Detection and Limit of Quantitation values were found to be 0.59µg/ml & 1.47µg/ml respectively. The result of the analysis for pharmaceutical formulation by the developed method was consistent with the label claim, highly reproducible and reliable. The method can be used for routine quality control analysis of Ampicillin Trihydrate in bulk and pharmaceutical formulations.

Conclusion
The UV spectroscopic AUC method for the analysis of Ampicillin Trihydrate by First order derivative Area under Curve was found to be simple, precise, and accurate; can be used for assay of bulk drug and pharmaceutical dosage formulations.