Developing Analytical Test Methods For the Simultaneous Quantitation of Actives, Preservatives & Impurities
Posted by Dr. Theo Kapanadze, D.Sc., Ph.D. on Tue, Jul 24, 2012
Developing and verifying a specific, accurate, precise and reproducible quality control method capable of simultaneously separating and quantifying actives, preservatives and impurities, including the separation of possible degradation products and excipients can be a complex and challenging task.
The combination of analytes in pharmaceutical formulations incorporating different ingredients that have very different polarity and therefore, chromatographic behavior can complicate developing an accurate reproducible analytical method. Moreover, the difficulty in their separation and analysis can be further complicated by the presence of impurities which can have very similar chemical structures. The dosage form may also contain excipients, some of which may interfere with the analysis of the active ingredients, primarily in the form of flavors and sugars which adsorb other compounds. Developing and validating a single method to determine all the ingredients quantitatively in combination can seem like an insurmountable task.
By using Ultra-Performance Liquid Chromatography/Ultra Violet Detection (UPLC/UV), a UPLC method with different chromatographic conditions for the separate and simultaneous quantification of actives, preservatives and their impurities can be achieved. UPLC methods can be more easily verified to be linear, reproducible, specific, sensitive, and rugged and can be adequate used for routine analysis of pharmaceutical formulations.
These methods can be suitable for identification, separation and quantitation of multiple actives from preservatives and other related substances. The high percentage of recovery that can be exhibited in these UPLC methods could indicate that the compounds are completely extracted from the formulations and the results indicate that the developed methods can be used to quantify all the listed analytes in these combinations without interference from other ingredients. Based on the results of the method verification phase based on system suitability, specificity, determination of QL and DL, linearity, accuracy and forced degradation studies, UPLC detection usually demonstrates an acceptable resolution. Because of superior resolution, the chromatographic conditions in these methods can show relatively good reproducibility of method performance with no significant interference peaks at the retention time of the various components.
UPLC methods can be successfully validated and used on a routine basis and can allow the quantitation of all analytes in pharmaceutical formulations in one single sample sequence run using three different gradient elution instrument methods in a short analytical time. Accuracy studies demonstrated that the methods exhibit reproducible and accurate results. These UPLC methods are sensitive, simple, use a fast, easy sample preparation procedure and possesses excellent linearity and precision characteristics.
Challenges are replicated whenever you are dealing with semisolid formulations, such as oral suspensions or topical products. Methylparaben and propylparaben are used as either single or in combinations in drug products as antimicrobial preservatives to prevent alteration of product preparations. Methylparaben is the methyl ester of p-hydroxybenzoic acid and propylparaben is the propyl ester of p-hydroxybenzoic acid.
Liquid preparations are particularly susceptible to microbial growth because of the nature of their ingredients. Such preparations are protected by the addition of preservatives that prevent the alteration and degradation of the product formulation.
The finished product release specifications should include an identification test and a content determination test with acceptance criteria and limits for each antimicrobial preservative present in the formulation. The finished product self-life specification should also include an identification test and limits for the antimicrobial preservatives present. This encourages the development of new stability-indicating methods for the simultaneous estimation of all compounds (API, impurities and preservatives) to provide a driving force in today’s pharmaceutical industry.
UPLC is a relatively new category of separation technique based upon well-established principles of liquid chromatography that utilizes sub-2 μm particles for the stationary phase. These particles operate at elevated mobile phase linear velocities to affect dramatic increases in resolution, sensitivity and speed of analysis. Owing to its speed and sensitivity, this technique has gained considerable attention in recent years for pharmaceuticals and biomedical analysis. In practice, this technology has been successfully applied to numerous method development and validation studies for assay determination in API, impurities and preservatives in solid, liquid and semisolid pharmaceutical formulations.
The main challenges for these methods are:
- Method should be able to determine the assay of all compounds in a single run
- Accuracy
- Reproducibility
- Robustness
- Stability-indicating
- Filter compatibility
- Linearity
- Free of interference from blank / placebo / impurities / degradation products
- Straightforward enough for routine use in a quality control laboratory
Developing methods in a short run time (in many cases less that 5 minutes) enables for rapid determination of drugs, impurities and preservatives. Also they can be utilized for determination of assay, blend uniformity and content uniformity of pharmaceutical products, where sample load is higher and high throughput is essential for faster delivery of results.
