2-picolylamine derivatization for high sensitivity detection of abscisic acid in apicomplexan blood-infecting parasites

Highlights

• A new mass spectrometry method for abscisic acid detection is proposed.

• The mechanism relies on picolylamine derivatization and positive ion mode detection.

• A 700-fold increase over commonly monitored underivatized abscisic acid is obtained.

• The abscisic acid limits of detection and quantification are 0.03 and 0.15 ng mL^-1.

• The method has been validated in the complex matrix of a red blood cell extract.

Abstract

We have developed a new liquid chromatography-electrospray ionization tandem mass spectrometry methodology based on 2-picolylamine derivatization and positive ion mode detection for abscisic acid (ABA) identification. The selected reaction leads to the formation of an amide derivative which contains a highly active pyridyl group. The enhanced ionization allows for a 700-fold increase over commonly monitored unmodified ABA, which in turn leads to excellent limits of detection and quantification values of 0.03 and 0.15 ng mL^-1, respectively. This method has been validated in the highly complex matrix of a red blood cell extract. In spite of the high sensitivity achieved, ABA could not be detected in Plasmodium falciparum-infected red blood cells, suggesting that, if present, it will be found either in ultratrace amounts or as brief bursts at defined time points within the intraerythrocytic cycle and/or in the form of a biosynthetic analogue.

Introduction

Human-infecting intracellular parasites of the Apicomplexa phylum are responsible for millions of clinical cases resulting in thousands of deaths every year, mainly in developing tropical regions [1]. The Apicomplexa with higher clinical impact can be classified into three principal genera/diseases: Plasmodium/malaria, Babesia/babesiosis and Toxoplasma/toxoplasmosis. All these pathogens share a characteristic organelle, the apicoplast, a non-photosynthetic plastid which has several metabolic pathways absent in humans and is therefore considered an exceptional target for antiparasitic drug research [2]. Isoprenoid metabolism operating in plastid-containing parasites has driven special attention because it is essential for the synthesis of numerous downstream metabolites that include ubiquinone, carotenoids and prenylation groups [3]. Carotenoid biosynthesis in the apicoplast has been described in Plasmodium falciparum [4] and in Toxoplasma gondii [5]. Considering the absence of photosynthetic activity in apicomplexan plastids, diverse alternative roles have been proposed for carotenoid derivatives in these parasites, including, among others [6], [7], [8], substrates for the synthesis of plant-like hormones that regulate essential cellular processes [5], [9], [10]. In this regard, abscisic acid (ABA, Fig. S1) has been proven to be endogenously produced by T. gondii through the C₄₀-carotenoid biosynthetic pathway (C₄₀-CBP) [5]. ABA is necessary for Toxoplasma reinvasion, regulating the maturation and egress of parasites from the host infected cell in a complex calcium-dependent signaling cascade [3]. The identification in P. falciparum of an host cell egress mechanism analogous to that present in T. gondii, together with the growth dependence of the parasite on intracellular oscillating calcium levels during most of its intraerythrocytic stage [11], are consistent with the possible existence of ABA or an ABA-like hormone in this pathogen. Accordingly, the herbicide fluridone (Fig. S1), which targets the phytoene desaturase enzyme in the early steps of the C₄₀-CBP, efficiently delayed T. gondii growth and prevented its multiplication while significantly reducing ABA levels in the intracellular parasite [5]. Other compounds with an even stronger effect on ABA synthesis, such as the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, Fig. S1), have been identified in plants [12] but to date have not been assayed against apicomplexan parasites.

Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is currently considered the technique of choice for the determination of ABA and other phytohormones [13], [14], [15], [16], [17], providing excellent sensitivity, specificity and throughput, and permitting the analysis of non-volatile and thermally unstable compounds. Although the sensitivity of LC-ESI-MS/MS (in the range of a few ng mL^-1) allows the detection of endogenous levels of ABA in plant material, significantly smaller concentrations of this hormone are expected to be produced by apicomplexan parasites due to the absence of most plant-like, ABA-regulated processes in these pathogens. This calls for the availability of high-sensitivity ABA detection protocols. Several chemical derivatization procedures have been developed in order to enhance the detection responses of carboxylic acid groups, such as the one present in ABA, in LC-ESI-MS/MS [18]. Picolylamine (PA), a derivatizing agent displaying a highly ESI-active pyridyl group that reacts with carboxylic acids in the presence of a condensation agent to form an amide derivative, has been employed for the analysis of fatty acids and other carboxyl-containing compounds [19].

In the present work we propose, for the first time, the use of PA for the derivatization of ABA as the basis of an ultra-high sensitive approach for the detection of this compound in red blood cell-infecting apicomplexan parasites, this being in turn a more efficient protocol for ABA determination than the currently employed gas chromatography-MS assay, which requires the methylation of ABA [5]. In addition to the optimized detection parameters, the results obtained after the application of a full validation protocol for ABA detection in P. falciparum are presented.