Determination of ampicillin in serum by using simple ultrafiltration technique and liquid chromatographic analysis.

This research describes a new liquid chromatographic method developed for detecting the antibiotic ampicillin in canine and equine serum, providing better recovery percentages and a lower detection limit.

Methodology

• The researchers have developed a new method to determine the presence and concentration of ampicillin in dog and horse serum using liquid chromatography.
• In this method, a 500 microL serum sample is mixed thoroughly with 500 microL of 20% ethanol. This mixture is then filtered using a microseparation tube, which has a 30,000 molecular weight cutoff. This allows larger molecules to be separated and filtered out during a low-speed centrifugation process.

Ampicillin Separation

• Following the initial filtration process, ampicillin is then separated from the other remaining components in the serum. This is achieved using a reverse phase ion-pair liquid chromatography (LC). In this process, nonpolar molecules are predominant leading to easy separation of ampicillin.
• The output from the liquid chromatography column is then examined using ultraviolet (UV) light at a wavelength of 230 nm. This UV absorbance allows the researchers to determine the amount of ampicillin in the serum sample.

Evaluation and Results

• The method was evaluated using dog serum spiked with ampicillin at concentrations of 10, 40, and 60 micrograms/mL. The recovery percentages of ampicillin, i.e., the percentage of ampicillin that could be successfully recovered from the serum were 93.1%, 100.9%, and 87.8% respectively.
• Similar evaluation was done using horse serum spiked with ampicillin at the same concentrations. The recovery percentages were 91.6%, 90.1%, and 88.7% respectively.
• The researchers also provided the coefficients of variation (CVs) values for the recovery of ampicillin from both dog and horse serum, which indicates the repeatability, or precision, of their method.
• The study found that the lower detection limit of ampicillin using this technique is less than 0.5 micrograms/mL serum.

Conclusion

• This research demonstrates the successful application of a new liquid chromatographic method for detecting the antibiotic ampicillin in canine and equine serum. This method provides promising high recovery rates and a low limit of detection, making it a potential candidate for detecting ampicillin in clinical settings.