The assay was successfully validation to current EMA guidelines. To the best of our knowledge the developed assay is the most sensitive published to date.

It is common practice to utilize an internal standard (IS) to minimize variance in bioanalytical assays employing liquid chromatography coupled to mass spectrometry. For assays to be deployed in regulated drug development studies, ensuring the IS will compensate for differences in recovery, liquid handling and ionization efficiency should be determined early in the method development process. In this perspective article, we outline key considerations when selecting an IS and propose experiments to perform within the method development phase to demonstrate suitability of the IS within the assay prior to validation. Finally, a series of case studies will be presented, which illustrate analytical challenges related to internal standardization that we have observed in our laboratory.

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The paper presents the validation and verification of an analytical method for the determination of total docosahexaenoic acid (DHA) in pig serum by liquid chromatography-electrospray ionization-tandem mass spectrometry. The characteristics studied during the validation included precision and accuracy, limit of quantitation (LOQ), selectivity, calibration range and linearity, parallelism, and stability. A separate verification study was also performed. The method was linear over the range. Precision and accuracy met acceptance criteria at all levels, and the LOQ was determined as 1 μg/mL. Parallelism experiments were conducted to show that there was no bias introduced in using a surrogate matrix to quantify DHA. Recoveries of free DHA were obtained for quality control samples, and stability studies were conducted over 1, 7, 31, and 180 days. The results of the verification study were in line with the validation study, and in conclusion, the method was deemed fit for purpose for measuring total DHA in pig serum.

Diets rich in omega-3 fatty acids (n-3 FA) have been associated with several health benefits. With the increased interest in n-3 FA both scientifically and societally, the accurate detection of such analytes has become increasingly important. Recently, tandem mass spectrometry (MS/MS) with electrospray ionization interface (ESI), hyphenated to both gas chromatography (GC) and liquid chromatography (LC), has become a valuable tool in the detection of docosahexaenoic acid (DHA). Liquid chromatography-electrospray ionization interface-tandem mass spectrometry methods have been developed for the determination of DHA in canine and poultry species. The objective of this article is to investigate whether LC-ESI-MS/MS is fit for purpose for the determination of DHA in laying hen serum. The disclosure of this work will be beneficial for researchers investigating poultry enrichment for regulatory and toxicological studies. The method was found to be linear over the range. Precision and accuracy results met acceptance criteria and the Limit of Quantitation (LOQ) was established as 1 µg/mL. Recoveries of DHA were obtained for quality control samples and stability studies were performed. The results of the verification study complimented those of the validation study. In summation, the method was established as fit for purpose for measuring total DHA in laying hen serum.