**1. Introduction**

Cardiac glycosides (CGs) are secondary metabolites produced by plants belonging to, among others, the genera *Nerium* (oleander)*, Convallaria* (lily-of-the-valley), and *Digitalis* (foxglove). The core structure of most CGs consists of lactone and steroid rings and a sugar moiety (Figure 1). CGs occur in all parts of plants and can be poisonous to livestock and humans. Their primary mechanism of action is inhibition of the membrane sodium-potassium pump that influences the intracellular sodium, calcium, and potassium concentrations and, as a consequence, causes disruptions in the cardiovascular system. However, other symptoms of toxicity may also include gastrointestinal, ocular, and neurologic disorders. In a specific dose range, however, CGs such as digoxin (DIGO) and digitoxin (DIGI) (Figure 1), have a long history of use as medications in treating various heart conditions [1–6].

In the literature, numerous cases of human poisoning with plants containing CGs through self-medication, accidental ingestion, suicide attempts, or criminal administration have been documented [7–11]. These reports also included a remarkable case of intoxication with CGs through food [8]. Superficial resemblance of the leaves of *Nerium oleander*, a plant producing toxic CG oleandrin (OLE), to the leaves of olive and bay trees (*Laurus nobilis*) might contribute to misidentification of the plant material and to accidental poisoning. Renal excretion is the main elimination route for some CGs (e.g., DIGO), while the hepatic route is more common to other CGs, combined with a partial renal elimination [12]. CGs excreted in urine are mainly unchanged [13–15] or partially metabolized [16].

A number of analytical, mostly single-analyte, methods for quantification of CGs in biological matrices have been described [11,17–24]. As a detection technique, these methods utilized mass spectrometry (MS) coupled to liquid chromatography (LC), which, thanks to its good selectivity and sensitivity, has nowadays become the method of choice for many applications including toxin analysis. Other techniques, such as immunoassay, (high-performance) thin-layer chromatography, and high-performance LC coupled to a UV or fluorescence detector [25–29], have also been applied to the detection and quantification of CGs.

Analytical methods for determination of these plant toxins in other than clinical samples are currently scarce. However, in case of a poisoning incident originating from the alimentary chain, the availability of a reliable method for food products is essential to confirm or rule out the ingestion of CG-containing plant material. Owing to the frequent use of herbal products as composite blends, and the complexity of the CG compound class, more than one CG might be associated with the poisoning, which points towards the significance of setting up multi-analyte methods. Therefore, the objective of this work was the development and validation of an ultra-high-performance (UHPLC)-MS/MS method for quantification of five plant toxins, namely OLE, DIGO, DIGI, convallatoxin (CON), and ouabain (OUB) (Figure 1), in edible herbs and spices and, complementary, in human urine. The choice of the target glycosides was dictated by their toxicity, known intoxication cases, occurrence of CG-producing plants, and availability of commercial reference standards.
