Due to the presence of endogenous components in biofluids, ionization suppression or enhancement may occur for bioanalytical assays using LC-MS or LC-MS/MS technologies. The matrix effect may affect the precision and accuracy of a bioanalytical method and, therefore, compromise the quality of the results. Protein precipitation sample preparation along with LC-MS/MS is a high-throughput method most commonly used in bioanalysis and is largely affected by the matrix effect. In order to eliminate the matrix effect during the method development, some considerations may be used: cleaner sample preparations, more sensitive instruments, which allow less material to be injected, different chromatographic separations and much more must be investigated. More than giving tools to adequately assess the matrix effect during the method development, this review gives scientists numerous ways to eliminate or reduce the matrix effect based on novel sample-preparation techniques, new chromatographic optimization methods and new technologies.

Analytical chemists should carefully choose the right combination of sample preparation and chromatographic conditions when working under HILIC conditions to avoid variable results.

Thiorphan, the active metabolite of racecadotril, can undergo oxidation in biological matrices such as blood and plasma. In bioanalysis, a general approach for the stabilization of such a molecule is to derivatize the thiol group to a more stable thioether, often requiring complex handling procedures at the clinical site. In this research, the concept of dried blood spot (DBS) on-card derivatization was evaluated to stabilize thiorphan. DBS cards were in-house pre-treated with 2-bromo-3'-methoxyacetophenone and left to dry prior to blood spotting. Thiorphan was shown to be effectively derivatized to thiorphan-methoxyacetophenone once applied on the in-house pre-treated cards. Thiorphan-methoxyacetophenone was extracted by soaking a 6 mm DBS punch in methanol containing the internal standard (thiorphan-methoxyacetophenone-D₅). Chromatographic separation was achieved on a Waters XBridge C₁₈ column with a gradient elution of 5 mM NH₄HCO₃ and methanol in 2.5 min and detection by ESI(+)/MS/MS. A linear (weighted 1/x²) relationship was obtained over a concentration range of 5.00-600.00 ng/mL. The assay met regulatory guidelines acceptance criteria for sensitivity, selectivity, precision and accuracy, matrix effect, recovery, dilution integrity and multiple stability evaluations. The DBS on-card derivatization has shown to be an easy and reliable alternative form of sample collection for the quantification of thiorphan.

Following this discovery, a reproducible method was developed and successfully validated for ramipril and ramiprilat. Additional stability tests were performed in the presence of glucuronide and diketopiperazine metabolites of ramipril and ramiprilat to demonstrate the method specificity.

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