One of the major problems associated with the therapeutic application of β-lactam antibiotics is their significant instability [1]-[3]. Analogs from that group are easily degraded in aqueous solutions and in solid state, which happens predominantly as a result of β-lactam ring chemical degradation. Because of this, the chemical integrity of β-lactam antibiotics must be closely monitored to meet FDA requirements. The original iodometric method based on visual detection of the end products of beta-lactamase hydrolysis that has been used to test beta-lactamase activity [4] was modified to quantitatively assess the amount of degraded product in amoxicillin. A UV-visible spectrophotometric method has been used, focusing on the ability of the products from the degradation of β-lactam analogs to absorb iodine from bulk solution, resulting in a significant reduction in the absorption spectra. Amoxicillin trihydrate tablets with different expiration dates and amoxicillin samples pre-loaded with known amounts of degraded amoxicillin have been used to generate a calibration curve and quantify the presence of degraded materials. Results show that the changes in the absorption spectra of iodine reagent directly correlate with the fraction of degraded β-lactam antibiotics, such as amoxicillin, in commercial products. The proposed assay might be as close as one step from simpler and cheaper methods of quantitative analysis of degraded β-lactam analogs, with estimated sensitivity in the range of 1% - 2%, which is better than the current FDA requirements.
β-lactam antibiotics have a broad spectrum of antibacterial uses and have such been successfully used in the treatment of a large number of diseases with bacterial etiology. The common element in the chemical structure of all β-lactam antibiotics is the presence of a five-membered β-lactam ring. Due to this underlying chemical specificity, they are easily degraded in both aqueous solution and in solid state [
One of the fastest and simplest methods for routine measurements of the integrity of the β-lactam ring of this group of antibiotics is based on the reduction of iodine by the hydrolyzed substrate [
In order to produce a quantitative positive control for β-lactam ring integrity, amoxicillin was hydrolyzed by alkaline hydrolysis [
Stock iodine solution was prepared by adding 0.7 g of I2 with 3.45 g of KI and dissolving it in 17.24 mL of distilled water.
Iodine reagent was prepared as described previously for the original fixed-time assay method [
A Lambda 35 UV-double beam spectrophotometer (Perkin Elmer Inc.) with a 1 mm length quartz cuvette was used for absorbance measurements.
Amoxicillin samples were prepared by the solubilization of amoxicillin powder (500 mg capsule) in 20 mM Na-P buffer, pH 7.0 at a final concentration of 2.5 mg/mL. Both a commercially available TEVA brand of amox- icillin and a commercially available Westward brand of amoxicillin were used for this study.
Degraded amoxicillin was prepared as follows, reproduced as according to [
To evaluate the degradation of amoxicillin in suspension over time, several identical suspensions were prepared and stored at room temperature (23˚C) in closed containers, each for a selected period of time. These samples were later assayed by iodometric titration using a spectrophotometric method.
solution resulted in a decrease of the absorbance at 350 nm due to the consumption of iodine by degraded amo- xicillin from a bulk solution (
It is interesting to note that amoxicillin degraded after a 20 min incubation period in 0.2 N NaOH solution, leading to the complete hydrolysis of the antibiotic [
However, the presence of degraded amoxicillin in a sample resulted in the reduction of iodine as described previously, leading to practically no changes in the absorbance at the spectrum peak at 350 nm, as the iodine added was consumed by the hydrolyzed amoxicillin. After the saturation point, the changes in absorbance increase linearly in a fashion similar to that of free iodine in solution. As follows from the plot, these two linear regions of the concentration-dependent function meet at a saturation point for the given concentration of amoxicillin in solution. For the sample presented, this point is reached after adding 40 μL of iodine reagent to the amoxicillin (concentration of 2.5 mg/mL).
Each data point was taken 3 times and then averaged to determine a statistically sound interval. The slope of each sample increases very slowly along the x-axis, as iodine reagent is added, until the saturation point is reached. From that point on, the added iodine acts like it does in free solution, contributing greatly to the slope of the sample. As is evident from the figure, the saturation point (the point at which there is no more degraded amoxicillin to react with the iodine reagent) correlates directly with the percentage of degraded material; given any future sample, a titration until the point of saturation would give us a clear measure of the substance’s integrity.
The stoichiometry of iodine molecules binding to molecules of degraded amoxicillin was determined from a titration of a 100% degraded amoxicillin solution with iodine reagent of a known concentration. Given an amoxicillin concentration of 2.5 mg/mL and a saturation point of 355 microliters of reagent, the molar ratio was calculated to be 1:6. This stoichiometry coefficient could then be used for quantitative analysis of amoxicillin samples with unknown fractions of degraded amoxicillin.
Using this knowledge, the calculations show that the concentration of degraded amoxicillin in our solution was 2.3 mg/mL. This was very close to the real concentration of the initially prepared amoxicillin solution, measured spectrophotometrically using the molar extinction coefficients ε274 mM = 1.4 and ε230 mM = 10.85. The small difference between these values may be associated with the kinetic factor of iodine discoloration.
In order to demonstrate the accuracy of the method, the titration was performed again with a smaller fraction of added iodine (5 μL, not shown). Based on the consistent distance between each slanted line, each representing an additional 5% of degraded material, we can interpolate based on the location of the fresh amoxicillin sample to determine that its integrity is mostly intact, with only 2% - 3% of degraded material. This in itself strikes an interesting observation; even amoxicillin that has not reached its expiration date contains some percent of degradation.
Since amoxicillin tends to degrade by a wide variety of chemical reactions, such as oxidation, photo-degrada- tion, and racemization, the effect of these reactions may interfere with the iodine titration assay. The yellowish color development of amoxicillin is generally considered as a visual criterion of the presence of degradation products. Therefore, we have tested how the development of a yellowish color of amoxicillin solution may interfere with the quantitative estimation of the β-lactam ring degradative material in a sample.
resized amount of the degraded amoxicillin in each sample was not freshly prepared, but instead stored over one week for the development of yellow color. As is the case with the other figures given, the saturation points seem to directly correlate with the percentage of degraded material. What is unique about this figure in particular are the rising starting absorbances, which then slope downward as iodine is added until the saturation point. The explanation for this phenomenon is that it is due to the discoloration of amoxicillin mentioned previously. However, this discoloration does not affect the integrity analysis method described. As iodine reagent is added, it reacts with the discolored degradation products and returns the absorbance to a semi-regular number, similar to as if the solution had not degraded over time at all; in essence, the saturation point is unchanged.
Together with the results presented in
Another find in this study suggests that the rate of degradation of amoxicillin in solution is substantially faster than determined in earlier studies. An increase by 5% in degraded products happens in the first day of storage at 23˚C vs. the 7 days found in previous studies [
In order to estimate the effect of ionic strength on iodine titration results, comparative measurements were performed in absence of and in presence of 0.1 M sodium chloride. Results show that for freshly prepared amo- xicillin solutions, the iodine titration curves are essentially the same for both the solution containing NaCl and the solution without. This clearly indicates that the interaction of iodine reagent with amoxicillin during titrations is unaffected by the presence of such a salt. Therefore, variations of ionic strength for a range of concentrations of NaCl do not affect the chemical reaction between iodine and the β-lactam ring of amoxicillin. In addition, the presence of NaCl in amoxicillin solution was found not to affect the rate of degradation for amoxicillin during storage at room temperature (data not shown).
The experimental results presented in this study demonstrate a potential for a practical application of the iodometric method, allowing for a fast and inexpensive evaluation of β-lactam antibiotics for impurities caused by β-lactam ring degradation.
This shows that freshly prepared samples made with amoxicillin past its expiration date contain more degraded material than do the freshly prepared samples from new amoxicillin. When tested in this study, the iodometric assay allows for the detection of β-lactam degraded material in amoxicillin commercial products, with an accuracy of ~2%. Our study also has indicated that freshly prepared amoxicillin suspensions made from 500 mg tablets of different manufacturers and taken prior to the expiration date may contain up to 3% of degraded amoxicillin.
In addition, development of a product assay using starch iodine as a color indicator could be based on the research done above. By comparing the intensity of a colored reaction (between β-lactam antibiotics and the starch iodine) to that of a quantitative standard, the percent of the degraded products in a stored container could be determined, similar to the technique previously developed based on an iodine-starch paper strip [
The research reported here was funded by the NASA Human Research Program (HRP), San Jose State University Bridges Program, and the NASA contract to the Carnegie Mellon University Silicon Valley # NNX08AB- 13A.
AbrahamSalois,IrisPerez,ErvinPalma,EthanGoolish,YuriGriko, (2015) Evaluation of the Chemical Integrity of Beta-Lactam Antibiotics by Iodine-Based Assay. Journal of Biosciences and Medicines,03,91-99. doi: 10.4236/jbm.2015.311012