Validated mass spectrometric assay for the quantification of substance P and human hemokinin-1 in plasma samples: A design of experiments concept for comprehensive method development

Abstract

IntroductionElevated plasma concentrations of the inflammatory neurokinins Substance P (SP) and human Hemokinin-1 (hHK-1) were found in infectious diseases. SP and hHK-1 plasma levels in diseased subjects are determined by immunoassays although not recommended by most immunoassay suppliers owing to their limitations to differentiate accurately between both peptides. A selective and reliable alternative (e.g. mass spectrometric (MS) assay) was missing because of a lack of sensitivity for the determination of endogenous plasma levels. MethodUsing a Design of Experiments (DoE) concept, a highly sensitive MS assay was developed for the quantification of SP, its inactive analog as the free acid, and hHK-1 in human plasma. Critical method aspects as the plasma extraction, peptide separation, and the method sensitivity were comprehensively optimized. The method was validated according to international bioanalytical guidelines and its applicability was evaluated in plasma of volunteers. ResultsWithin 106 experiments utilizing the DoE concept, the sensitivity of the assay was substantially improved to achieve limits of detection of 5.8 pg/mL for SP, 6.2 pg/mL for its free acid, and 5.3 pg/mL for hHK-1 in plasma. The lean method development was followed by the successful validation according to the regulatory guidelines resulting in a wide quantification range of 7.8–2000 pg/mL. In the volunteers’ plasma, no SP and hHK-1 were detectable. Instead, the free acid of SP was quantified in individually distinct levels (202.5–1024.1 pg/mL). ConclusionAn accurate and precise MS assay for the quantification of SP, its free acid, and hHK-1 in plasma was established. The mass spectrometric quantification of the free acid of SP in human plasma samples lead to the question about possible cross-reactivity of the immunoassays in former determinations.