## 1 Introduction  
The compounds 3,4-dihydroxypropylglycol (DHPG), 3-methoxy-4-hydroxyphenylglyglycol (MHPG), and vanillymandelic acid (VMA) are metabolic products of epinephrine (E) and norepinephrine (NE), which are part of the catecholamine (CA) group. These compounds are produced through the enzymatic actions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT). After metabolism, they are released into the cerebrospinal fluid (CSF) and blood (specifically, plasma), and ultimately excreted from the body through urine. Their elevated levels in the blood are linked to stress and may result from excessive production of catecholamines by neuroendocrine tumor cells or be associated with certain mental health disorders [1, 2]. DHPG is formed from NE by deamination to dihydroxyphenylglycolaldehyde (DOPEGAL) and reduction of the aldehyde by aldehyde reductase. Subsequently, DHPG is metabolized by COMT to form MHPG. An elevated NE:DHPG ratio in patients, could be the symptoms of inhibition activity of MAO or of aldehyde/aldose reductase [3]. Decreased DHPG levels are observed in [PERSON] disease [4], which affects infants and is a fatal neurodegenerative X chromosome-linked recessive disease. In turn, patients with depression resistant to dexamethasone exhibit increased excretion of DHPG [5]. The concentration levels of MHPG and DHPG in urine samples can be influenced by various factors, including age, gender, diet, health status, the use of medications and psychoactive substances, as well as the techniques used for sample collection and storage. Consequently, reference values for MHPG and DHPG in urine may vary depending on the laboratory and the testing methodology employed. MHPG, a major metabolite of norepinephrine (NE), serves as a biomarker for evaluating nervous system function. It is used in the clinical diagnosis of various diseases, including Parkinson's disease, vascular parkinsonism, depression, schizophrenia, mental disorders, bipolar disorder, and acute stroke [6; 7; 8; 9; 10; 11; 12; 13]. The literature suggests that MHPG can serve as a marker in the diagnosis of pheochromocytomas or paragangliomas, as MHPG secretion from pheochromocytomas is significantly higher than from normal tissues [14; 15]. MHPG is metabolized to VMA in the liver by the action of alcohol dehydrogenase. Measuring VMA in a 24 h urine sample is valuable for diagnosing pheochromocytoma and neuroblastoma [16]. VMA deficiency is linked to a congenital deficiency of DOPA decarboxylase, the enzyme responsible for converting DOPA to dopamine [17]. In patients with known hypertension, urinary VMA levels can be more than seven times higher, and in those with pheochromocytoma, the levels may exceed those in healthy individuals by over 35 times. The determination of catecholamines (CAs) and their metabolites has evolved from early bioassays, colorimetric, and fluorometric methods to liquid chromatography-based techniques for measuring these compounds in plasma and urine [18]. Liquid chromatography with electrochemical detection (LC-ECD) or coupled with tandem mass spectrometry (LC-MS/MS) is among the most commonly used analytical techniques [19; 20; 21; 22]. Immunoassays have also gained popularity for measuring plasma catecholamine metabolites; however, despite their high throughput, immunoassay suffer from limitations such as low sensitivity and specificity [23; 24]. High-performance methodologies often require labor-intensive sample preparation and long chromatographic run times to minimize potential interferences that may co-elute with CAs. Consequently, there is still a need for rapid, efficient, and specific methods to isolate analytes from biological matrices. While most studies have focused on plasma samples, 24 h urine samples, blood, serum, and human and rat brain tissue have also been investigated [25; 26; 10; 27]. Liquid-liquid extraction (LLE) has been widely used for isolating CA metabolites from urine. This method often involves the use of organic solvents to separate the analytes from the aqueous phase.