**1. Introduction**

Histochrome® is a solution of the sodium salt of naturally occurring quinone echinochrome A (7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone) for intravenous injections and infusions, manufactured in ampoules (Supplementary Materials, Figure S1). Histochrome is registered in

Russia as an antioxidant drug. It is used in cardiology for the treatment of coronary heart disease and for restriction of the necrosis zone in myocardial infarction (state registration number P N002363/01), and in ophthalmology for the treatment of dystrophic diseases of the retina and cornea, macular degeneration, primary open-angle glaucoma, diabetic retinopathy, hemorrhages to vitreous humor, retina, and anterior chamber, and discirculatory disorders in the central artery and retinal vein (state registration number P N002363/02). Histochrome has no known analogues, and it simultaneously blocks a number of free-radical reactions, neutralizes reactive oxygen species (ROS), nitric oxide, and peroxide radicals, chelates metal ions, inhibits lipid peroxidation, and regulates antioxidant enzyme levels [1].

Echinochrome A (Ech A, **1**) is one of the main pigments of various sea urchin species [2–4] (Figure 1). Ech A isolated from the sand dollar *Scaphechinus mirabilis* (purity >98%) is registered in the Russian pharmacopeia as an active drug substance under the international non-patented name pentahydroxyethylnaphthoquinone (state registration number P N002362/01).

**Figure 1.** Chemical structure of echinochrome A (7-ethyl-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone, Ech A).

Currently, more and more papers focused on elucidating the mechanisms underlying the diverse biological effects of Ech A are being published. Ech A was found to protect rat cardiomyoblasts and isolated cardiomyocytes from the effects of cardiotoxic compounds doxorubicin, *tert*-butyl hydroperoxide, and sodium nitroprusside, which cause an increase in ROS formation and depolarization of mitochondrial membranes [5]. In rat cardiomyoblast cells, Ech A dose-dependently increased the mass of mitochondria and the content of mitochondrial DNA and activated mitochondrial biogenesis, increasing the expression of the main regulators of the metabolic function of mitochondria [6]. Ech A was also found to activate mitochondrial biogenesis in skeletal muscle, increasing the endurance of rats during physical activity by increasing the number of mitochondria [7]. Being an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2<sup>+</sup> ATPase 2a (SERCA2A) receptor, which is responsible for pumping calcium ions from the cytosol into the sarcoplasmic reticulum, Ech A prevented ischemic damage of the myocardium, reducing the area of myocardial infarction [8]. By reducing the level of intracellular ROS and regulating the expression of pro- and antiapoptotic proteins, Ech A protected human cardiac progenitor cells against oxidative stress [9]. This may be the basis for a simple and effective strategy to enhance myocardial regeneration by increasing the survival of transplanted cardiac cells under oxidative stress induced by ischemic damage. Ech A was found to be an effective agent for promoting cell proliferation and maintaining the stemness of hematopoietic stem and progenitor cells [10]. Ech A is also beneficial for human CD34+ progenitor cells from peripheral blood to maintain their self-renewal potential and function during ex vivo expansion. The efficacy of Ech A in a model of hemorrhagic and ischemic stroke in rats has been demonstrated [11,12]. It was found that the drug can cross the blood–brain barrier into the cerebrospinal fluid. Ech A also exhibited an antidiabetic effect due to antioxidant and hypoglycemic activities [13,14]. A study demonstrated that Ech A inhibits acetylcholinesterase and exhibits dose-dependent antiradical activity against nitric oxide, which opens up its possible use in the treatment of neurodegenerative diseases [15]. The therapeutic potential of Ech A in the treatment of various inflammatory diseases has been demonstrated in numerous studies: in a model of experimental colitis in mice [16], in an experimental model of bleomycin-induced pneumonia in immature rats [17], in children aged 7–12 years old with chronic inflammatory lung diseases [18,19], and in adolescents with erosive gastroduodenitis [20,21].

Since the search for active compounds and the creation of new drugs is a long, expensive, and risky process, one of the main modern pharmaceutical strategies is the use of registered drugs for a new medical application. Considering the above variety of activities possessed by Ech A with an established mechanism of action, the development of new dosage forms based on this substance seems promising.

To ensure the quality of active pharmaceutical substances and finished drug products, impurities must be monitored carefully during process development, optimization, and changeover. The isolation, characterization, and control of impurities in pharmaceutical substances are being reviewed with a greater focus on national regulatory and international guidelines [22]. According to International Conference on Harmonization (ICH) guidance Q3A(R2) and Q3B(R2), degradation products are impurities resulting from a chemical change in the drug substance during manufacture and/or storage of the drug product due to the effect of light, temperature, pH, water, or reaction with an excipient and/or the immediate container closure system. Due to the presence of a large number of phenolic hydroxyls, Ech A readily undergoes oxidative decomposition. The aim of this study was to isolate and determine the structure of Ech A degradation products formed during the oxidation of its preparation (Histochrome) by O2 in air-equilibrated aqueous solutions.
