Analytical Method Development and Validation of Assay of Ampicillin Trihydrate Using UV-Visible Spectroscopy

Ampicillin Trihydrate is a semi-synthetic, broad-spectrum β-lactam antibiotic that belongs to the penicillin group. It is extensively used to treat a variety of infections caused by both Gram-positive and Gram-negative bacteria, including Streptococcus, Staphylococcus, Escherichia coli, Haemophilus influenzae, and Neisseria gonorrhoeae. This antibiotic is effective against respiratory tract infections, urinary tract infections, gastrointestinal infections, meningitis, and sepsis. Ampicillin works by inhibiting bacterial cell wall synthesis, ultimately leading to the weakening of the cell wall and the death of the bacterial cell.[4][5][6][7]

The term "Trihydrate" in ampicillin refers to the presence of three water molecules of crystallization associated with each drug molecule. This crystalline hydrate form is crucial for determining the compound's physical and chemical stability. Compared to the anhydrous form, the trihydrate form is more stable, less hygroscopic, and easier to handle. The water molecules in its crystal lattice help maintain the drug's structural integrity, flow properties, and shelf life. Additionally, they enhance solubility and dissolution characteristics, which are essential for achieving optimal bioavailability and therapeutic effect upon administration.[1]

In pharmaceutical formulations, Ampicillin Trihydrate is commonly used in capsules, tablets, and dry syrup preparations for oral administration. Ensuring the purity, potency, and uniformity of ampicillin in these dosage forms is vital for maintaining quality control and therapeutic efficacy. Therefore, analytical methods that are simple, precise, and accurate are required for its routine analysis.[9][10]

Among various analytical techniques, UV-Visible spectroscopy is one of the most preferred due to its simplicity, cost-effectiveness, and rapidity. It measures the absorbance of ultraviolet light by the drug at a specific wavelength (λmax), which can be directly correlated to its concentration using Beer-Lambert’s law.[2][3]

Several reported methods used different solvent systems such as methanol, water, phosphate buffer, and their mixtures. Based on the comparative evaluation of previous studies, a diluent containing methanol and water in the ratio of 60:40 v/v was optimized to achieve maximum solubility, clarity, and reproducibility of absorbance for ampicillin trihydrate. This optimization ensured the best balance between solvent polarity and drug stability, providing a reliable medium for further analytical validation.[8][11][12]

Thus, the objective of the present study is to develop and validate a simple, accurate, and reliable UV spectroscopic method for estimating Ampicillin Trihydrate in bulk and pharmaceutical dosage forms, in accordance with ICH Q2(R2) guidelines. This method aims to ensure the quality assurance and standardization of Ampicillin Trihydrate formulations in pharmaceutical analysis.[12][13][14].

Figure 1: Structure of Ampicillin Trihydrate

MATERIALS AND METHOD:

MATERIALS:

Ampicillin Trihydrate (API), Ampicillin Trihydrate Capsules, Methanol, Distilled Water

Instruments:

Analytical balance (Shimadzu), UV-Visible spectrophotometer (Shimadzu-1800).

METHOD:

Preparation of Diluent:

The solvent employed for the analysis consisted of a blend of methanol and distilled water at a ratio of 60:40 (v/v), ensuring ideal solubility and transparency of the Ampicillin Trihydrate solution for UV spectroscopic assessment.

Preparation of standard stock solution:

Accurately weighed and transferred 5.0 mg of Ampicillin Trihydrate Working Standard into a 100 ml volumetric flask added a sufficient quantity of diluent and Sonicated to dissolve. Made up the volume to the mark with the diluent.

Determination of Maximum Wavelength (λmax):

A standard solution of Ampicillin Trihydrate was scanned in the ultraviolet range of 200–400 nm using a UV–Visible spectrophotometer, with the prepared diluent serving as the blank. The absorption spectrum obtained exhibited a well-defined peak at 219 nm, which corresponds to the maximum absorbance of the drug. This wavelength (λmax) was selected for all further quantitative measurements and method validation studies, as it represents the point of highest sensitivity and accuracy for Ampicillin Trihydrate analysis.

Analysis of Ampicillin Trihydrate in capsule Formulation:

The capsule formulation contains 250 mg of Ampicillin Trihydrate in a total capsule weight of 500 mg. The average weight of the capsules was found to be 600 mg. Based on this, the equivalent weight of capsule powder required for analysis was calculated as 12 mg to correspond to the actual drug content. The accurately weighed capsule powder was transferred into a 100 mL volumetric flask and dissolved in a diluent. The solution was mixed thoroughly to ensure complete dissolution of the drug. The absorbance of the solution was measured against a blank solution (diluent only) using a UV-visible spectrophotometer at the previously determined λmax of 219 nm. The concentration of Ampicillin Trihydrate in the capsule formulation was calculated using the calibration curve prepared from the standard stock solution.

Validation:

The developed UV-spectrophotometric method was validated as per ICH Q2(R2) guidelines. Parameters such as linearity, accuracy, precision, limit of detection, and limit of quantitation were evaluated. The method showed excellent linearity within the selected concentration range with acceptable accuracy and precision. These results confirm the reliability and suitability of the method for routine analysis of Ampicillin Trihydrate.^[14]

Linearity:

The linearity of Ampicillin Trihydrate was evaluated by preparing eight different concentrations ranging from 6 to 300 µg/mL from the standard stock solution. These solutions were analyzed at the previously determined λmax of 219 nm using a UV-visible spectrophotometer. The absorbance of each concentration was measured against a blank solution (diluent only). A calibration curve was constructed by plotting absorbance versus concentration, and the linearity was assessed by calculating the correlation coefficient (R²). The results demonstrated a direct and proportional relationship between absorbance and concentration within the studied range, indicating that the method is suitable for quantitative determination of Ampicillin Trihydrate in both pure form and capsule formulation.

Table 1: Results of Linearity

Figure 2: Calibration curve for Ampicillin Trihydrate (Conc. vs. Abs.)

Range:

Range is an interval between highest and lowest concentration limit of the analyte i.e. 6-          300µg/ml.

Accuracy:

The concentration 25, 50, 75 µg/ml was taken as 50%,100%,150% and % recovery was found to be in range 99%-101%. Hence the parameter was found to be validated.

In precision intra-day and inter-day precision were performed at concentration (50µg/ml). The obtained results were found within limit i.e. less than 2%RSD.

Ruggedness (Intermediate Precision):

The change in analyst with same concentration and environmental condition didn’t affect the results.

Table 6: Comparative Results of (Intermediate Precision) Ruggedness

Robustness:

The change in Diluent from 60:40 ml v/v to 70:30ml v/v and 50:50 ml v/v didn’t affect the results.

The assay was performed by using Ampicillin Trihydrate Capsules at concentration 50µg/ml. The % purity was found to be 99.73%.