Analytical Methods for Identification and Quantification of Quinoa Saponins: A Review

Abstract

Quinoa saponins (SAPs) are key secondary metabolites occurring as complex mixtures mainly in the seed coat of Chenopodium quinoa Willd. Although traditionally removed due to their bitter taste and potential toxicity, quinoa SAPs display diverse biological activities, including anti-inflammatory, hypocholesterolemic, antifungal, molluscicidal, hemolytic, and cytotoxic effects, that support their potential applications in pharmaceuticals, functional foods, cosmetics, and biopesticides. Their amphiphilic nature also enables their use as natural emulsifiers. This review (1981–2024) summarizes advances in analytical methodologies for quinoa SAPs, emphasizing that while GC-MS and LC-MS/MS are widely applied for profiling, full structural elucidation still requires isolation and analysis by NMR and MS. We discuss key considerations for quinoa SAPs identification using GC-MS, LC-MS/MS, and NMR. Quantification remains challenging and is often based on relative estimations, with afrosymmetric, UV–vis, and GC-MS methods being the most frequently employed, while HPLC-DAD, LC-MS, and GC-MS/MS offer greater sensitivity. Ultimately, the selection of the analytical method and standard critically determines accuracy.

1. Introduction: General Aspects and Properties of Quinoa Saponins

1.1. General Aspects of Quinoa

Quinoa (Chenopodium quinoa Willd.) is a food plant domesticated in the Andes of South America (Peru and Bolivia), with archeological evidence about 5000–7000 years ago, and clearer evidence of its agronomic development between 1500 B.C. and 100 A.D. in the southern Lake Titicaca basin (Bolivia).

Quinoa is a plant of great adaptability, and it is a drought tolerant crop being able to develop in regions with very low annual precipitation (200–400 mm), but it can also be grown in regions with high annual precipitation (3000 mm), like in southern Chile. In addition, it has high salinity tolerance, unlike other crops like rice or maize. With climate change increasing dry, saline areas and a growing population, stress-tolerant, nutritious crops are crucial for future food security. Quinoa, known for its resilience and excellent nutrition, has gained significant attention.

Studies show quinoa’s nutritional superiority over grains like rye, rice, barley, and oats due to its higher quantity and quality of nutrients. For example, quinoa contains more and higher-quality proteins with a balanced pattern of essential amino acids, including lysine, methionine, and threonine, which are limited in cereals like wheat and maize. , The carbohydrate content is similar to that of wheat and rice, with starch being the major one (32–69%). In addition, quinoa grains have an interesting lipid composition (5.5–7.4 g/100 g edible matter), made up mainly of unsaturated fatty acids, such as oleic, linoleic, and alpha-linolenic, which represent 88% of the total fatty acids. Additionally, it has a high concentration of vitamin E, vitamin C, riboflavin (B₂), pyridoxine (B₆), and folic acid, giving quinoa important functional properties. The mineral content of quinoa is also of great importance; the seeds have bioavailable forms of calcium, magnesium, iron, and potassium, with good proportions suitable for a balanced diet. Additionally, quinoa is a gluten-free food being a healthy option for human consumption.

Quinoa’s resilience to abiotic stress and its beneficial properties have driven its global expansion. While Peru and Bolivia remain the top producers, quinoa is now cultivated on every continent, with growing production in countries like the USA, Canada, Chile, Ecuador, Colombia, Denmark, India, and China. , In addition, currently there is a growing interest in quinoa as a functional food, because in addition to its high content of proteins, lipids, fiber, vitamins, and minerals, and its excellent balance of essential amino acids, quinoa contains important quantities of bioactive phytochemicals like saponins, phytosterols, phytoecdysteroids, phenolic compounds, polysaccharides, bioactive proteins, and peptides. Recent investigations have demonstrated the beneficial effects of these compounds on metabolic, cardiovascular, and gastrointestinal health, leading to their increased recognition as a functional food. , Many studies have confirmed the antioxidant, hypolipidemic, immunomodulatory, and weight-regulating effects of the quinoa diet, contributing to liver and cardiovascular health. In addition, some research has reported the hypoglycemic, hypotensive, probiotic, antitumor, and hormone-regulating potential of quinoa, but more data are required make conclusions about these functions. Considering all this, we can say that quinoa can be considered as a healthy and functional food.

1.2. Quinoa Saponins Generalities

Saponins (SAPs) are nonvolatile glycosidic secondary metabolites synthesized by a significant number of plant families, found in cultivated and wild plants like quillaja, quinoa, sugar beet, oats, soybean, legumes, ginseng, asparagus, and alfalfa. A common structural characteristic of SAPs is the presence of a triterpene or steroid aglycone, which is typically linked to one or two oligosaccharide groups through ether or ester bonds. The combination of hydrophilic sugar groups and hydrophobic aglycones (also known as sapogenins) imparts an amphiphilic character to these compounds, which gives many SAPs notable surface activity. This amphiphilic character is probably correlated with the biological activities of SAPs.

The quinoa SAPs are one of the major and most characteristic phytochemicals in C. quinoa. Chemically, quinoa SAPs are found in a complex mixture of mono- and bidesmosidic SAPs; although there are some reports mentioning tridesmosidic SAPs in quinoa, just one (quinoside A) was identified as tridesmosidic SAP. Monodesmosidic SAPs possess a single oligosaccharide chain attached normally at C-3; bidesmosidic SAPs are the most common quinoa SAPs, these triterpene glycosides consist mainly in a hydrophilic oligosaccharide linked to hydrophobic triterpene by an ether bond in C-3 and normally a glucose bound to the aglycone by an ester bond in C-28. The common sugars are glucose (Glc), galactose (Gal), arabinose (Ara), glucuronic acid (GlcA), and xylose (Xyl), and the sapogenin is usually oleanolic acid OA (1), hederagenin HD (2), phytolaccagenic acid PA (3), or serjanic acid SA (4), but spergulagenic acid (5), gypsogenin (6), 3-β-hydroxy-27-oxo-olean-12-en – 28-oic acid (7) and 3-β,23-α,30-β-trihydroxy-olean-12-en-28-oic acid (8) as quinoa sapogenins were also reported (Figure ).

1.

Structures of triterpene sapogenins identified in Chenopodium quinoa Willd.

The triterpene SAPs have been found in all parts of the quinoa plant such as leaves, flowers, fruits, seeds, and seed coats , but in the grains are normally concentrated in the seed coat. Their presence in crops is undesirable for food and feed due to their bitter taste, antifeedant properties, and hemolytic effects, classifying them as antinutritional factors requiring removal to enhance sensory quality and edibility. , For this, several methods are used to remove most of the SAPs from the grains, including wet, dry, and genetic methods. Among these methods, the scarification combined with water washing is one of the most common methods used at the industrial level. The process begins with dry mechanical scarification, where grains are rubbed among them to remove the seed coat, followed by a wet washing step to eliminate remaining bitter impurities, but demanding less water than in processes where only wet methods are used.

Quinoa saponins (SAPs) are often labeled as antinutritional factors, but this term applies only to compounds that reduce a food’s nutritional value by hindering nutrient digestion, absorption, or use. While saponins can cause hemolysis in direct blood contact, their toxicity is minimal when ingested. Preclinical in vivo studies found no adverse effects at doses below 50 mg/kg of body weight per day. Additionally, some SAPs form complexes with iron or zinc, but there is no evidence they form complexes with vitamins. Then, there is very limiting evidence to say that quinoa SAPs are antinutritional factors, and moreover they showed toxic effects only in high concentrations. Some in vivo studies showed that as triterpenoid SAPs with amphiphilic activity, they can stimulate the digestive tract at high doses (more than 10 g per kg); in addition, they demonstrated that the excretion time of quinoa SAPs in the body is long, and they have limited acute toxicity effects as a plant active ingredient, and the results of the mutagenicity experiments showed that quinoa SAPs had no mutagenesis in mice. Quinoa SAPs could then be used in a new type of product, but other toxicological studies are still needed to confirm the safety in long-term consumption. Nevertheless, their elimination in quinoa grains before consumption is due more to their bitter test than their possible toxicity.

SAPs content in quinoa depends on the genotypes. Considering their SAPs content, quinoa can be classified as sweet genotypes (free from or containing <0.11% of free saponins, around of 20 to 40 mg/100g dry weight) or bitter genotypes (containing >0.11% of free saponins, around of 140 to 2300 mg/100 g dry weight). ,, Because of their bitter taste, and possible antinutritional factors, in some countries there is a maximum acceptable level of quinoa SAPs for human consumption (free containing <0.12%).

1.3. General Biological and Pharmacological Properties of Quinoa Saponins

The role of SAPs in quinoa plants is not fully understood, although they are considered to serve as a defense mechanism against harmful microorganisms, as well as other threats such as birds and insects. , Their biopesticidal potential was first demonstrated by San Martn and colleagues, who identified a possible application as a molluscicide against Pomacea canaliculata (Golden Apple Snail), a species that causes economic damage to rice crops worldwide. They reported that alkali-treated quinoa husks killed 100% of the snails within 24 h at 33 ppm, while showing no toxicity to fish up to 54 ppm. ,

Quinoa SAPs also have antifungal effects. While SAPs crude fractions moderately inhibited Candida albicans, alkali treatment enhanced activity by converting bidesmosidic SAPs into monodesmosidic forms, which more effectively can dive into biomembranes with their lipophilic side chain, while their hydrophilic oligosaccharide chain interacts with extracellular glycoproteins and glycolipids, causing alterations of biomembranes and lysis at high concentrations. Supporting this, Stuardo and San Martin demonstrated that at doses of 5 mg/mL of alkali treated quinoa SAPs have a 100% of conidial germination inhibition in Botrytis cinerea, while nontreated SAPs had no effects. , Similarly, antifungal activity of an extract rich in quinoa SAPs against Cercospora beticola was evaluated showing good activity from 5 mg/mL. However, purified quinoa SAPs showed little or no antifungal activity, suggesting a synergistic effect among them, Furthermore, the carbohydrate chain bonded to C-3 was shown to be critical for the antifungal activity of SAPs, as its removal often results in loss of activity.

The first biopesticide/fungicide based on quinoa SAPs was commercialized based on a patent of Dustcheshen. The product “Heads Up” plant protector can be applied as a suspension on tubers, legumes, and cereal seeds, to prevent fungal, bacterial attacks, and viral plant diseases; however, little research is available on its efficacy.

From a toxicological perspective, monodesmosidic SAPs exhibit higher hemolytic activity than bidesmosidic ones due to their stronger ability to lyse biomembranes. The hemolytic activity seems to be dependent on the number of monosaccharides linked to C-3, as well as on the functional groups of the triterpene sapogenin; moreover, the presence of a free carboxylic group at C-28 seems to be essential for their cytotoxicity. Nevertheless, some reports indicate that the hemolytic activities are not necessarily linked to the cytotoxicity. In addition, despite their hemolytic properties, limited evidence suggest direct toxicity of quinoa SAPs in nonaquatic animals, although respiratory damage has been seen in fish.

Given the global concern over obesity and dyslipidemia, quinoa SAPs have attracted attention for their potential effects on lipid metabolism. In this context, quinoa SAPs were related to a decrease in weight gain and levels of cholesterol, triglycerides, low-density lipoproteins (LDL), and high-density lipoproteins (HDL). For example, the inhibitory activity of SAP-rich quinoa extracts and their hydrolysates was evaluated in vitro against pancreatic lipase. Both exhibited inhibitory activity, with hydrolysis enhancing the effect and additionally reducing bioaccessible cholesterol. Similarly, supplementation with quinoa SAPs in hyperlipidemic rats reduced serum lipids, body weight, liver injury, and inflammation, thereby preventing lipid metabolism disorders. Pasko et al. (2010) reported that quinoa seeds reduced serum total cholesterol (26%), LDL (57%), glucose (10%), and plasma total protein (16%), in high-fructose-fed rats, suggesting that the hypocholesterolemic effect could be attributed to fiber, SAPs, and/or squalene. However, the effect of quinoa grains to reduce the levels of cholesterol, triglycerides, low-density lipoproteins (LDL), and high-density lipoproteins (HDL) seems to be due to different compounds, like proteins, SAPs, fiber, and phytosterols, − not only to SAPs, since the hypocholesterolemic effect persisted with desaponified grain meal in rat studies.

The cytotoxic and apoptotic activities of quinoa SAPs have also been explored. For instance, two ethanolic extracts from white and red quinoa agro-industrial residues as well as three sapogenins were tested against cancer cell lines (A549, SH-SY5Y, HepG2, and HeLa). While the extracts showed no significant cytotoxicity, the sapogenins OA (1) and HD (2) displayed strong activity. Kuljanabhagavad et al. (2008), studied the cytotoxic effect on 20 quinoa SAPs employing HeLa cells and MTT assay, determining activity in two bidesmosidic saponins (34) and (35) both with SA (4) as sapogenin. Quinoa SAPs have also shown promise as immunological adjuvants. In mice, quinoa-derived SAPs enhanced both systemic and mucosal antibody responses when used as mucosal adjuvants. Moreover, the cytotoxicity of four isolated quinoa sapogenins (1), (2), (3), and (4) (Figure ) was evaluated in one normal-like human breast epithelial cell line (MCF-10A) and in one breast cancer cell line (JIMT-1) using an MTT dose response assay, determining that HD (2) showed interesting cytotoxicity being more toxic in JIMT-1 cells (IC₅₀ 27.3 μM) than in MCF-10A normal cells (IC₅₀ 39.6 μM). Finally, in another study, OA (1) showed cytotoxicity against the following lines: prostate cancer PC3 cells (IC₅₀ 6.5 μM), lung cancer A549 cells (IC₅₀ 0.4 μM), breast cancer MCF-7 cells (IC₅₀ 35.4 μM), and gastric cancer BGC823 cells (IC₅₀ 2.6 μM).

Other important activity thoroughly investigated in quinoa SAPs is its anti-inflammatory activity. Yao et al. (2014) evaluated four quinoa SAP fractions, reporting reduced nitric oxide (NO) production and inhibition of inflammatory cytokines such as TNF-α and IL-6. In another study, a sapogenin extract and four isolated sapogenins (OA, HD, PA, and SA) were tested by using mouse models of ear and paw edema. The extract showed higher anti-inflammatory activity than the isolated compounds in the ear edema model, suggesting a synergistic effect, whereas in the paw edema model the isolated compounds were more effective. Based in these studies, among the pharmaceutical applications, quinoa SAPs could have anti-inflammatory uses.

Regarding their biological role in plants, the localization of SAPs on the outer layer of quinoa seeds suggests a role in plant development and germination, similar to that observed in Avena spp. (oats) and Vicia sativa, but there are no studies for quinoa SAPs. Tolerance to abiotic stresses, such as high salinity, could be an important biological role of quinoa SAPs, but there is not a clear explanation for this role. − However, two experiments were performed to improve the germination under salinity using SAP seed priming, finding that SAP seed priming mitigates the negative effects of salt stress, suggesting that it could be used as an easy and cost-effective technology for quinoa crop growth on saline soils. Additionally, they can have a potential function as allelopathic agents, but all of these biological actions need further studies.

Analyzing all of the quinoa SAPs properties (Figure ) and considering that SAPs are often removed from quinoa before consumption, due to their unpalatable taste, giving a byproduct rich in these biological active compounds, various applications have been proposed. Potential uses include their incorporation into cosmetic or pharmaceutical preparations , as well as an active component for biopesticide products or functional food. Their amphiphilic nature also makes them attractive as natural emulsifiers in food or cosmetic formulations. As a functional food factor, quinoa SAPs are valuable as additives and in medical care. Finally, it is important to emphasize that toxicological studies in rats showed limited acute toxicity and no mutagenicity in mice, supporting their safe and rational use.

2.

Properties of quinoa saponins.

1.4. General Aspects of Quinoa Saponins Analysis

For the analysis of quinoa SAPs, it is important to consider that they represent a large group of secondary metabolites with highly diverse structures. The eight oleanane-type aglycones (sapogenins) can be substituted with one, two, or three sugar chains, yielding mono-, bi-, or tridesmosidic SAPs, respectively, with numerous monosaccharide variations. To date, more than 90 different SAPs have been identified in quinoa seed hulls, although only about 30 have been isolated. However, in quinoa extracts, SAPs occur as a complex mixture of compounds with similar polarities and structural features, making their separation, analysis, and identification particularly challenging.

SAPs are identified by different techniques, among them we have the less precise TLC analysis by spraying with sulfuric acid or a mixture of p-anisaldehyde-sulfuric acid-glacial acetic showing blue-violet spots or using vanillin as a reagent to react with the hydroxyl groups at C-3, but not all the SAPs are detected; also it is common to use their surfactant properties to give a stable foam formation to detect quinoa SAPs, see section 4.1.2, in addition is common to detect quinoa SAPs using their hemolytic activity, see section 4.1.6, and finally there are colorimetric reactions, like Liebermann Burchard sensitive to UV–vis for spectrophotometric detection, which will be explained more in the quantitative analysis but it is also used for qualitative detection of quinoa SAPs (see Section 4.1.4). All the mentioned techniques are simple tests which could be used for a rough detection of quinoa SAPs, but for more precise results, instrumental techniques such as HPLC-DAD, LC-MS, LC-MS/MS, GC-MS or NMR can be used.

One of the main challenges in analyzing quinoa SAPs by UV–vis or chromatographic methods such as HPLC-DAD, LC-MS, LC-MS/MS, or GC/MS is the matrix effect. In the extracts, quinoa SAPs occur in a complex mixture together with other compounds, including glycosylated flavonoids, phenolics, and sugars, which can interfere with the analysis. For example, quinoa SAPs exhibit poor UV absorption because they lack chromophore groups in their structure, making them less sensitive in HPLC with UV detectors and restricting their detection to λ ≤ 220 nm. At this wavelength, other compounds present in the complex matrix, such as phenolics, can also absorb, leading to overlapping signals and difficulties in detection and quantification. Several strategies have been proposed to minimize this matrix effect, including improved extraction methodologies that combine sample extraction with analytical preparation steps, such as solid phase extraction (SPE) or liquid–liquid extraction (LLE). These approaches reduce extraction time and the number of processing steps, ultimately improving sample quality. Traditional separation methods, such as the use of macroporous resins, are time-consuming and provide low accuracy, whereas SPE has been shown to reduce matrix effects effectively. In addition, SPE can be applied for sample cleanup and for preconcentration of SAPs in matrices with low abundance, making it particularly useful prior to LC-MS quantification of quinoa SAPs.

NMR spectroscopy is a unique technique to determine the exact structure of quinoa SAPs because the MS techniques cannot determine the stereochemical configurations that are crucial for the identification of sugars. But, for NMR analysis, it is necessary to have isolated SAPs, and isolation is usually time-consuming and requires a large amount of sample, which also means money. In this sense, NMR spectroscopy is considered unsuitable for the identification of quinoa SAPs in a mixture of SAPs, so, nowadays, chromatographic techniques coupled to mass spectrometry are increasingly favored to identify SAPs, since they do not need isolated SAPs, and their efficiency in identifying compounds within complex mixtures is sufficient for most quinoa SAPs studies.

The structural determination of individual SAPs presents challenges owing to their structural intricacy; it usually requires the combination of several techniques like GC-MS, LC-MS/MS, and NMR. Moreover, several researchers reported the use of hydrolysis reactions for the analysis in some techniques (like GC-MS) or just to simplify the structural analysis. Figure shows the different types of hydrolysis that could be used considering that quinoa SAPs are glycosides in which the hemiacetal hydroxyl groups of saccharides form acetals with a triterpene residue.

• i. Hydrolysis acid or complete hydrolysis of a glycoside. The glycoside bond is cleaved, breaking both ether and ester linkages, which releases the monosaccharides and the aglycone. However, since SAPs are fragile molecules, acid hydrolysis frequently generates artifacts. For this reason, control of the reaction time is very important usually 1 to 3 h. In some works, the reaction is stopped in a very short time to obtain the pro-sapogenins that have undergone partial hydrolysis, which are purified and analyzed by TLC and NMR.
• ii.Through basic hydrolysis, the ester bond that unites the glucose with the sapogenin is broken. In quinoa SAPs, the cleavage of O-acylglycosidic sugar chains in basic hydrolysis conditions typically is carried out by refluxing with 5% potassium hydroxide ,,,− or 5% aqueous NaOH or 0.5 M potassium hydroxide for 2 h. , Alternatively, the use of 1 to 20% ethanolic or methanolic solutions of potassium hydroxide is possible, but there is a risk of methylation, especially of the carboxyl group of triterpene acids.
• iii. Enzymatic hydrolysis is a very effective and gentle method for the cleavage of sugar residues from SAPs without artifact formation. Mizui et al. (1988) used crude hesperidinase in a quinoa study obtaining rhamnose residue in this hydrolysis. In other studies of isolation and structural identification used α-glucosidase (maltase, Sigma Type III) and β-glucosidase (emulsin, Sigma Type II) to confirm the configuration for α or β linkage. ,

3.

Types of quinoa SAPs hydrolysis methods.

Developments in equipment for the separation and identification of molecules from new organisms are leading to the increasing isolation of SAPs. Normally obtaining pure SAPs has used combinations of adsorption chromatography (TLC and CC) and gel filtration (Sephadex), and finally it is usual to use preparative HPLC. However, the quantities of isolated pure SAPs are usually small because SAPs extracts are complex and very sensitive methods that have high resolution and are nondestructive normally are necessary to help the structural determination.

To report the molecular structure of quinoa SAPs, advances in nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) have been essential. Thus, FAB-MS provides information on the molecular weight, and although it is destructive it only requires less than one mg to provide information on the SAPs such as the sequence of sugars. Mono- and two-dimensional NMR techniques allow for the bonds between sugars and contribute to the determination of the structure of the aglycone.

The quantification of quinoa SAPs can be done by different methods, but it is not possible to have the exact quantification of quinoa SAPs; normally, we have only a relative estimated value based on some properties of these compounds. There are several techniques available, with UV–vis and GC-MS being the most frequently employed. For any quantification, it is very important to have a proper standard; in this sense, it is possible to use a purified fraction of quinoa SAPs or a pure SAP preferably obtained from quinoa as a standard. Pure sapogenins, such as OA, can also serve as standards for indirect SAPs quantification; however, the choice of standard depends on the method chosen for quantitative analysis. The UV–vis methods are not available to direct detection of SAPs because they do not have a chromophore group, so they are all performed using colorimetric assays; among them, the Liebermann-Burchard and its modified method are the simplest and most adopted methods because they are not very expensive and time demanding. For more precise quantification, the most commonly reported methods are LC-MS and GC-MS.

Since quinoa SAPs are gaining attention due to their interesting biological, pharmaceutical properties, and potential industrial uses, correct selection of extraction methods and analytical techniques based on a correct literature review is needed. In this review, we will describe the extraction methods of quinoa SAPs and the combined chromatographic and spectroscopic techniques for the qualitative and quantitative analysis of quinoa SAPs trying to facilitate future work on identification and quantification of quinoa SAPs.

2. Extraction Methods of Quinoa Saponins

Several methods for the extraction of SAPs are found in the literature. ,− Ethanol and methanol as well as the hydroalcoholic mixtures are still considered the most effective solvents for the extraction of quinoa SAPs by conventional methods, due to the high solubility of these compounds in such solvents. However, these extracts also contain other compounds, like phenolic compounds and sugars; for this, in order to obtain an extract enriched in SAPs, the extraction is normally followed by a partition between n-butanol and water where the main part of quinoa SAPs remain in the n-butanol phase. Nevertheless, some reports have shown that the aqueous phase contains some quinoa SAPs that are not present in the butanol phase. Other studies initially perform defatting with petroleum ether and then perform an extraction with polar solvents such as methanol or ethanol or hydroalcoholic mixtures; for example, Woldemichael and Wink (2001) used a Soxhlet methanol extraction for 72 h of defatted quinoa grains, The solvent was then removed by distillation under reduced pressure to obtain the solid extract, which was suspended in water and partitioned with n-butanol, where the SAPs are usually recovered.

On the other hand, some researchers used other nonconventional methods to improve the efficiency of extraction reducing the extract time and the quantity of solvents; among them we can mention the extraction of quinoa SAPs using microwaves. In a study of Gianna et al. (2012), the authors evaluated the effect of different parameters such as temperature, seed/solvent ratio, treatment time, and % of alcohol as solvent; the results showed that the optimal extraction conditions were using ethanol/water and isopropanol/water 20%, 20 min at 90 °C. The extraction of quinoa SAPs using microwaves reduced the extraction time, and the use of alcohol as a solvent makes it possible to extract more quinoa SAPs than using water alone. Another study showed the extraction of quinoa SAPs using pressurized hot water extraction (PHWE) in an accelerated solvent extraction (ASE 350) equipment obtaining very good results working at 195 °C in 1 min. Anyway, the conventional extraction methods using hydroalcoholic mixtures are still preferred by the researchers of quinoa SAPs. ,

3. Identification and Structural Elucidation of Quinoa Saponins

3.1. Identification and Structural Elucidation by NMR

As we mentioned above, isolation and purification of quinoa SAPs are complicated processes and are time-consuming because the extracts are complex mixtures of individual SAPs with several similar features and polarity. A cascade of separation techniques like different types of chromatographic methods must be used, and it is common to use combined techniques to determine the structure.

For example, if we get an isolated SAP, the unique way to determine the exact structure will be using 1D and 2D NMR techniques. But even when we get an individual SAP the analysis using NMR techniques presents several challenges due to its structural complexity. So, the ¹H NMR spectrum of a pure SAP usually shows a lot of overlapped signals in the whole spectrum, in one way due to the aliphatic protons of the triterpene side and in other way due to the many protons of carbons substituted by hydroxyl groups. Therefore, to determine the structure we need, ¹³C NMR and also 2D NMR experiments give more information about the ¹³C NMR tables to arrive at structure identification than ¹H NMR tables. For example, the ¹³C NMR chemical shift values give specific and important information about the substituents, on the oleanane skeleton, in C-3, C12, C13, C-28, C-30, C-24, and C-27, which is very useful information for identification (Figure ). Many researchers who studied the components of quinoa SAPs used acid hydrolysis to obtain sapogenins (triterpenes). Burnouf-Radosevich et al. (1984) reported the isolation and identification of seven triterpenes type oleanane as sapogenins in C. quinoa by normal phase HPLC on a silica gel column. In other study, Burnouf-Radosevich et al (1985) reported nine trimethylsilylated pentacyclic triterpenes by gas chromatography, and thus OA (1) and HD (2) were confirmed to be major triterpenes of quinoa seeds. A review by El Hazzam et al. published in 2020 reported that eight sapogenins were found in quinoa (see Figure ), but only for six of them is it possible to find the ¹³C NMR data in the bibliography, and the major ones are OA (1), HD (2), and PA (3). Table shows the ¹³C NMR data of the six sapogenins of Chenopodium quinoa Willd.

4.

Example of ¹³C NMR of a quinoa SAP obtained with 500 MHz Bruker equipment (own source).

1. ¹³C NMR Data (δ) of Chenopodium quinoa Sapogenins.

C	OA (1)	HD (2)	PA (3)	SA (4)	Spergulagenic acid (5)	Gypsogenin (6)
1	36.5	38.9	38.7	38.9	38.9	38.4
2	26.9	27.6	27.6	28.1	28.1	27.0
3	76.7	73.7	73.5	78.1	78.1	71.6
4	37.9	42.9	42.7	39.4	39.4	56.2
5	54.7	48.8	48.6	55.9	55.9	48.0
6	17.9	18.7	18.6	18.8	18.8	21.0
7	32.0	33.0	32.9	33.3	33.3	32.5
8	38.3	39.8	39.7	39.7	39.7	40.0
9	47.0	48.2	48.1	48.1	48.1	47.7
10	33.2	37.3	37.2	37.4	37.4	36.2
11	22.5	23.8	23.8	23.8	23.8	23.8
12	121.4	122.7	123.2	123.2	123.2	122.2
13	143.7	145.0	144.4	144.5	144.5	144.8
14	41.2	42.2	42.1	42.1	42.1	42.2
15	27.1	28.4	28.4	28.4	28.4	28.2
16	22.8	23.8	23.8	23.9	23.9	23.8
17	45.6	46.7	46.1	46.2	46.2	46.6
18	40.7	42.0	43.3	43.4	43.4	41.9
19	45.3	46.5	42.7	42.7	42.7	46.5
20	30.3	31.0	44.2	44.2	44.2	30.9
21	32.8	34.3	30.8	30.9	30.9	34.2
22	30.6	33.3	34.5	34.5	34.5	23.1
23	28.1	68.2	68.0	28.8	28.8	207.1
24	15.9	13.1	13.1	16.5	16.5	9.6
25	15.0	16.0	15.9	15.5	15.5	15.7
26	16.8	17.5	17.4	17.4	17.4	17.3
27	25.5	26.2	26.1	26.1	26.1	26.1
28	178.5	180.4	179.1	179.9	179.9	180.0
29	32.3	33.3	28.4	28.4	28.4	33.2
30	23.4	23.8	177.1	177.2	177.2	23.8
OCH3			51.7	51.7

The structural identification of the quinoa SAPs mixtures, as we indicated, is very complex and tedious, if not impossible. , For this, due to the number of compounds present in the samples, one can focus on one of the major compounds, which involves the non-negligible issue of choosing the molecules of interest and therefore requires a priori knowledge. However, this approach typically requires compound separation and purification, which consumes time, money, and human resources.

In contrast, NMR fingerprinting aims to establish a global approach: the mixture is subjected to the NMR experiment as a whole. A simple quantification of the main compounds or compounds of interest can be carried out. For this, one or more signals must be identified in the NMR spectrum. The NMR spectra of complex mixtures show, like SAPs, hundreds of signals from numerous compounds. This and the overlapping signals make it difficult to extract information, either “visually” or through simple data processing. The analysis of these “global profiles” is carried out through the use of chemometric tools that allow identifying the characteristic patterns of the molecules of interest.

Nevertheless, in some cases, it was possible to get pure quinoa SAPs. The following Tables – show the compilation of reported ¹³C NMR data of the isolated SAPs in different organs of C. quinoa which were compiled in order to contribute to the construction of these global profiles as well as to make easy the identification of other isolated quinoa SAPs using NMR techniques.

2. ¹³C NMR Data (δ) of Hederagenin HD (2) Saponins Isolated in Chenopodium quinoa ,,,

6. Main Fragments of Silylated Sapogenins Observed in GC-MS Analysis.

individual sapogenins (A)			silylated sapogenins (B)
Aglycone	R1	R2	R1	R2	principal fragments of silylated sapogenins
Oleanolic acid OA (1)	CH3	CH3	CH3	CH3	600 (molecular ion, M•+; 3.5%), 482 (M•+ – TMSCOOH; 21.4%), 320 (M•+ – ABC rings rDA1, 40%), 203 (M•+ – ABC rings rDA1 – TMSCOO•; 100%) (Figure )
Hederagenin HD (2)	CH2OH	CH3	CH2OSi(CH3)3	CH3	688 (molecular ion, M•+; 3.2%), 598 (M•+ – TMSOH; 3.3%), 570 (M•+ – TMSCOOH; 14.5%), 320 (M•+ – ABC rings rDA1; 42.2%), 203 (M•+ – ABC rings rDA1 – TMSCOO•; 100%).
Serjanic acid SA (4)	CH3	COOCH3	CH3	COOCH3	644 (molecular ion, M•+), 629 (M•+ – •CH3), 526 (M•+ – TMSCOOH), 364 (M•+ – ABC rings rDA1), 247 (M•+ – ABC rings rDA1 – TMSCOO•)
Phytolaccagenic acid PA (3)	CH2OH	COOCH3	CH2OSi(CH3)3	COOCH3	732 (molecular ion, M•+; 1.2%), 642 (M•+ – TMSOH; 1.9%), 614 (M•+ – TMSCOOH; 11%), 364 (M•+ – ABC rings rDA1; 21.5%), 247 (M•+ – ABC rings rDA1– TMSCOO•; 85%)

Table shows the ¹³C NMR data of HD-SAPs (Hederagenin SAPs) that were isolated from C. quinoa. Thus, Woldemichael and Wink reported two monodesmosidic SAPs derived from the basic hydrolysis with hederagenin as sapogenin: hederagenin 3-O-α-l-arabinopyranoside (9) and hederagenin 3-O-β-d-glucopyranosyl-(1,3)-α-l-arabinopyranoside (10), but no ¹³C NMR data were reported by Woldemichael and Wink. In Table the ¹³C NMR for (9) was obtained from Li et al. (2015) and for (10) from Jhosi et al. (1992). Furthermore, both monodesmosidic SAPs showed strong activity in a hemolytic test compared with their bidesmosidic form (11) and (12), see Figure , and in addition Li et al. 2015 found that the monodesmosidic SAPs hederagenin 3-O-α-l-arabinopyranoside (9) and pulsatilla saponin A (similar to 10) isolated from Hedera nepalensis inhibited lung cancer A549 cell growth in a dose-dependent manner, through a mechanism including induction of cancer cell apoptosis.

5.

Hederagenin HD (2) saponins of Chenopodium quinoa.

Tree bidesmosidic HD-SAPs from quinoa bran were isolated by Mizui et al. (1988), hederagenin 3-O-α-l-arabinopyranosyl-28-β-O-d-glucopyranoside (11), hederagenin 3-O-β-d-glucopyranosyl-(1,3)-α-l-arabinopyranosyl-28-O-d-glucopyranoside (12), and hederagenin 3-O-β-d-glucopyranosyl-(1,3)-α-l-galactopyranosyl-28-O-β-d-glucopyranoside (13) with their respective ¹³C NMR data and Miziu et al. (1990), besides (11), (12), (13) reported the ¹³C NMR data of hederagenin 3-O-β-d-glucuronopyranosyl-28-O-β-d-glucopyranoside (14). and hederagenin 3-O-β-d-xylopyranosyl-(1,3)- β-d-glucuronopyranosyl-28-O-β-d-glucopyranoside (15), and finally Dini et al. (2001) besides (12) reported the hederagenin 3-O-β-d-glucopyranosyl-(1,4)-β-d-glucopyranosyl-(1,4)-β-d-glucopyranosyl-28-O-β-d-glucopyranoside (16) with their respective ¹³C NMR data (see Table ). Later, the isolation of these SAPs were reported by Woldemichael and Wink. (2001), Kuljanabhagavad et al. (2008), Dini et al. (2002), and Zhu et al. (2002).

Woldemichael et al. in 2001 reported the 12 SAPs in C. quinoa, and one of them was oleanolic acid 3-O-β-d-glucuronopyranoside (17). This SAP was reported as a prosapogenin, a product of basic hydrolysis of SAP (22), and also in 2002 the team of Zhu et al. reported the isolation of SAP (17) in C. quinoa, but they did not report the ¹³C NMR data for SAP (17). The chemicals shift shown in Table were obtained of the work of Kizu et al. (1985) that studied Hedera nepalensis.

3. ¹³C NMR Data (δ) of Oleanolic Acid OA (1) Saponins Isolated in Chenopodium quinoa ,,− .

Ma et al. (1989) reported the isolation of four OA-SAPs from C. quinoa: hederoside A2 or oleanolic acid 3-O-β-d-glucopyranoside (18), oleanolic acid 3-O-β-d-xylopyranosyl-(1,3)-β-d-glucuronopyranoside (19), oleanolic acid 3-O-β-d-xylopyranosyl-(1,3)-β-d-glucuronopyranosyl 28-O-β-d-glucopyranoside (20), and oleanolic acid 3-O-β-d-xylopyranosyl-(1,3)-β-d-glucuronopyranosyl-6-methyl-ester 28-O-β-d-glucopyranoside, but did not report ¹³C NMR data, and thus the ¹³C NMR data of (18) were obtained from the work of Miyakoshi et al. (1999) that isolated this SAP from Acanthopanax nipponicus. For SAP (19) the ¹³C NMR chemical shifts were obtained from Espada et al. (1990), who isolated the SAPs from Boussingaultia baselloides and for SAP (20) the ¹³C NMR data were obtained from Mizui in 1990. Mizui et al. (1990) studied C. quinoa and reported 14 SAPs, and three of them are OA-SAPs quinoside D (20), oleanolic acid 3-O-β-d-glucopyranosyl-(1,2)-β-d-glucopyranosyl-(1,3)-β-arabinopyranosyl-28-O-β-glucopyranoside (21), and oleanolic acid 3-O-β-d-glucuronopyranosyl-28-O-β-d-glucopyranoside (22) (see Figure , Table ).

6.

Saponins of oleanolic acid OA (1) as sapogenin isolated in Chenopodium quinoa.

Dini et al. (2001) reported the isolation of six SAP of C. quinoa: two of them were SAPs of OA: oleanolic acid 3-O-α-l-arabinopyranosil-(1,3)- β-d-glucuropyranosyl 28-O-β-d-glucopyranoside (23) and oleanolic acid 3-O-β-d-glucopyranosyl-(1,3)-a-l-arabinopyranosyl 28-O-β-d-glucopyranoside (24). Dini reported their ¹³C NMR spectra for these SAPs.

The SAP oleanolic acid 3-O-β-d-glucuronopyranosyl-28-O-β-d-glucopyranoside (22) is denominated Chikusetsusaponin IVa, , and it has some pharmacological properties as an activator of protein kinase and provides resistance to thrombus and some metabolic diseases. Yin et al. (2018) and Xu et al. (2023) reported that SAP (22) inhibited endometrial carcinoma cell proliferation and the cell cycle, and furthermore confirmed that SAP (22) stimulated the apoptosis of endometrial carcinoma (EC) cells. Therefore, SAP (22) could serve as a potential drug for EC treatment.

From C. quinoa seed, Woldemichael and Wink (2001) reported the isolation of several SAPs, of which three of them had PA (3) as sapogenin: phytolaccagenic acid 3-O-β-d-glucopyranosyl (1,3)-α-l-arabinopyranoside (25), phytolaccagenic acid 3-O-α-l-arabinopyranosyl 28-O-β-d-glucopyranoside (26) and phytolaccagenic acid 3-O-β-d-glucopyranosyl-(1,3)-α-l-arabinopyranosyl-28-O-β-d-glucopyranoside (27). In 2001, Dini’s team found two SAPs among others with PA (3) as sapogenin: phytolaccagenic acid 3-O-[α-l-arabinopyranosyl-(1,3)-β-d-glucuronopyranosyl]-28-O-β-d-glucopyranoside (28) and phytolaccagenic acid 3-O-β-d-glucopyranosyl-(1,4)- β-d-glucopyranosyl-(1,4)-β-d-glucopyranosyl-28-O-β-d-glucopyranoside (29). Finally Mizui et al. (1988) reported the isolation of phytolaccagenic acid 3-O-β-d-galactopyranosyl-(1,3)-β-d-glucopyranosyl 28-O-β-d-glucopyranoside (30). Figure shows the structure of PA-SAPs, and Table shows their ¹³C NMR data.

7.

Phytolaccagenic acid PA (3) saponins isolated in Chenopodium quinoa.

4. ¹³C NMR Data (δ) of Phytolaccagenic Acid PA (3) Saponins in Chenopodium Quinoa ,,

In addition, from C quinoa it was possible to isolate SAPs that were made up of other sapogenins, and thus Mizui et al. (1990) reported the isolation of spergulagenic acid 3-O-β-d-glucopyranosyl-(1,2)-β-d-glucopyranosyl-(1,3)-a-l-arabinopyranosidese (31). Sapogenin serjanic acid SA (4) is a methyl ester of spergulagenic acid (see Figure ), Dini et al. (2001) reported the isolation of two SAPs of spergulagenic acid, but analyzing the ¹³C NMR data, they were SA-SAPs: serjanic acid 3-O-β-d-glucopyranosyl-(1,2)-β-d-glucopyranosyl-(1,3)-a-L-arabinopyranosyl-28-O-β-d-glucopyranoside (32) and serjanic acid 3-O-[a-l-arabinopyranosyl-(1,3)-β-d-glucuronopyranosyl]-28-O-β-d-glucopyranoside (33). Kuljanabhagavad et al. (2008) reported the isolation of several quinoa SAPs and presented ¹³C NMR data of serjanic acid 3-O-a-l-arabinopyranosyl-28-O-β-d-glucopyranoside (34) and serjanic acid 3-O-β-d-glucuronopyranosyl-28-O-β-d-glucopyranoside (35). Finally, Dini et al. (2002) also reported the identification of a new saponin: serjanic acid 3-O-β-d-glucopyranosyl-(1,3)-a-l-arabinopyranosyl-28-O-β-d-glucopyranoside (36), and the characterization based on spectroscopic and chemical data indicated that the carbon that had carboxylate was C-29 and not C-30 of sapogenin. In addition to compounds (34) and (35), Kuljanabhagavad et al. (2008) also reported ¹³C NMR data for two other SAPs: 3β-[(O-β-d-glucopyranosyl-(1,3)-α-l-arabinopyranosyl)­oxy]-23-oxo-olean-12-en-28-oic acid β-d-glucopyranoside (37) and 3β-[(O-β-d-glucopyranosyl-(1,3)-α-l-arabinopyranosyl)­oxy]-27-oxo-olean-12-en-28-oic acid β-d-glucopyranoside (38). Figure shows the structures of several SAPs isolated from quinoa that present minor sapogenins, and Table shows their ¹³C NMR data.

8.

SAPs with spergulagenic acid (5) and serjanic acid SA (4), 3-β-hydroxy-23-oxo-olean-12-en-28-oic acid (= gypsogenin) (6) and 3-β-hydroxy-27-oxo-olean-12-en-28-oic acid (7) as quinoa sapogenins.

5. ¹³C NMR Data (δ) of SAPs of Spergulagenic Acid (5), Serjanic Acid SA (4), Gypsogenin (6), and Other Minor Sapogenins from Chenopodium quinoa ,,,

3.2. Identification by GC-MS

Numerous methodologies have been employed for the identification of SAPs; however, as we said, the determination of individual SAPs presents challenges owing to their structural intricacy and intricate separation processes. Consequently, NMR spectroscopy is deemed unsuitable for the identification of quinoa SAPs, primarily due to the time-intensive purification steps involved. Then, nowadays, chromatographic techniques coupled with mass spectrometry are increasingly favored to identify SAPs, for their efficacy in identifying compounds within complex mixtures.

Gas chromatography coupled to mass spectrometry (GC-MS) with electronic impact (EI) has been used for identification of quinoa aglycones (sapogenins) and is one of the most used options for indirect SAPs determination and identification. However, a very important requirement for GC-MS analysis is the volatility of the molecule, which is an inconvenient for direct SAPs analysis. SAPs have many hydroxyl groups in the sugars linked to the triterpene structure which allows its volatilization. That is why SAPs must be hydrolyzed and then derivatized by formation of trimethylsilyl volatile derivatives prior to GC-MS injection. One of the most used reagents to obtain trimethylsilyl derivatives is the N,O-bis­(trimethylsilyl)­trifluoroacetamide trifluoroacetamide (BSTFA) (Figure ).

9.

Silylation reaction of quinoa sapogenins. A) Oleanane sapogenins (1–4) and B) silyl derivatives of sapogenins (1–4).

The MS detector shows all structural information on sapogenins identification obtained from MS fragments analysis, ,, where the main peaks identified correspond to the fragmentation of silylated sapogenins starting with the elimination of TMS, formic acid and formaldehyde that is shown in Table .

The most important signals correspond to the molecular ion at m/z 600, at m/z 482 by loss of the TMSCOOH group and at m/z 393 by loss of the TMSO^• and TMSCOOH groups. The most intense signals at m/z 279 and 203 are formed by the retro Diels–Alder (rDA) reaction (Figure ). However, minor fragmentation occurs by rupture of the pentacyclic ring ABC. ,,, This method is not only used for identification means but also for quantitative analysis, as we can see in Section 4.2.5.

10.

Typical GC-MS fragmentation of oleanane triterpenoid.

3.3. Identification by LC-MS and LC-MS/MS

Liquid chromatography coupled to mass spectrometry (LC-MS) and GC-MS are the most used options for determination and identification of quinoa SAPs. The wide array of SAP structures present in quinoa represents a significant challenge for researchers, prompting the need for novel and sensitive techniques. However, this challenge can be addressed by employing liquid chromatography coupled with mass spectrometry. It has been reported different ionization techniques used in LC-MS and LC-MS/MS, in order to identify SAPs in quinoa such as atmospheric pressure chemical ionization (APCI) and electrospray Ionization (ESI) in positive and negative modes. APCI in positive and negative modes give more useful information about the number of SAPs present in the extract, but ESI in positive mode shows more information about sapogenins than in negative mode.

Madl et al. (2006) utilized tandem LC-MS/MS employing nanospray ionization with an ion trap and GC-MS to report 87 quinoa SAPs. Their study provided comprehensive preassignments and identification of eight types of sapogenins, along with elucidating the sequence of saccharide groups joined to aglycones. Additionally, Segura et al. (2020) employed a combination of MALDI-MS, LC–MS, and LC–MS/MS to identify the elemental composition of 14 SAPs in Chilean quinoa extracts, discerning their sapogenins, the number and type of sugars, and confirming that the saccharide attached to C-28 is always glucose. Furthermore, Ruiz et al. (2017) identified approximately 24 SAPs in quinoa seeds using LC-MS/MS, employing tandem MS analysis, and GC-MS. Their findings revealed HD (2) as the most abundant aglycone, followed by PA (3) and OA (1), with sapogenin determination primarily conducted via GC-MS. By LC-MS/MS, they elucidated the aglycone molecular weights and typical fragmentation of saccharide portions, which occurs by losing hexoses, with a molecular weight of 162 Da and an adduct ion with mass of 172 Da, which could come from the acyl moiety.

LC-MS and LC-MS/MS are the most common techniques for SAPs identification recorded in positive and negative ionization mode, where the typical ions can be observed such as (M + H)⁺ or (M + H – H₂O)⁺, because SAP has a labile hydroxyl group, which join with the signals observed due to the loss of the saccharide groups.

As an example to show how it is possible to identify SAPs using LC-MS/MS, we are going to take two quinoa SAPs, phytolaccagenic acid 3-O-β-d-glucopyranosyl-(1,3)-α-l-arabinopyranosyl-28-O-β-d-glucopyranoside (27) and oleanolic acid 3-O-β-d-glucuronopyranosyl-28-O-β-d-glucopyranoside (22) described by Madl et al. (2006). For identification, they used two steps of analysis. First, the aglycones were identified by GC-MS analysis of hydrolyzed products, and second, successive LC-MS and nLC-MS/MS analyses were used to distinguish the aglycone, the saccharides, and to determine the oligosaccharide sequence and branching. In the LC-MS/MS analysis, they observed the following:

• i)The molecular weight was detected as [M + Na]⁺ or [M + H]⁺ for (27) (m/z 995 and 973) and for (22) (m/z 817 and 795); in the case of the presence of a sugar acid like in (22) [M⁺² Na-H]⁺ (m/z 839) was observed.
• ii)[M + H – Glc]⁺ ion, for 27 (m/z 811) and for 22 (m/z 633), which was generated at higher ionization voltages, was selected for an MS/MS spectrum showing in all the compounds the loss of the C-28 esterified monosaccharide residue. This result is supported by quantum chemical calculations which show that bond breaking between the C-28 carboxylic group and the monosaccharide is energetically favored with respect to fragmentation at the C-3 bound saccharide.
• iii)The MS/MS of [M + H – 192]⁺ provided information for the sequence of the oligosaccharide chain linked at C-3. So, for (27) losses of a hexose (m/z 649) and pentose (m/z 517) residues were detected, according to Glc-Ara-linked to the C-3, while for (22) it showed the fragment consistent with the loss of GlcA (m/z 457).
• iv)The loss of saccharides produced ions specific for the aglycones, such as PA (m/z 517) for (27) and OA (m/z 457) for (22).
• v)After the loss of all 28-O and 3-O linked saccharides, the charge is located at the C-3 hydroxyl group favoring the water elimination resulting in a strong signal at m/z 499 for 27 and at m/z 439 for 22. For PA, the aglycone comprises an additional hydroxyl group giving the elimination of two water molecules (m/z 481). For all the compounds, water elimination was accompanied by neutral loss of formic acid (46 μ) due to the C-28 carboxylic acid.

Figure shows the scheme of fragmentation of compound (27) (phytolaccagenic acid 3-O-[β-d-glucopyranosyl-(1,3)-α-l-arabinopyranosyl]-28-O-β-d-glucopyranoside) as an example of the structural elucidation using LC-MS/MS. But one important thing to take into account is that MS analysis cannot give the exact structure because the stereochemical information is not available by MS.

11.

Example of MS fragmentation of a saponin (27) using tandem mass spectrometry (nLC-ESI-MS/MS).

Table shows the ionization methods and some of the principal fragments reported using LC-MS/MS techniques for quinoa SAPs.

7. Principal Fragments Using Different Ionization Methods in the LC-MS/MS Analysis of Quinoa SAPs .

ionization	principal fragments	information	analytical method and references
ESI and APCI (+)	[M + Na]+; [M + K]+; [M + H]+	Molecular weight	nLC-ESI-MS/MS
	[M + 2Na-H]+	Sugar acid	LC-ESI-MS/MS and LC-APCI-MS/MS
	[M + H-162]+ at high ionization voltage	Glc at C-28
	MS/MS of [M + H-162]+	Oligosaccharide chain linked at C-3
	[Agl + H-H2O]+ strong peak	Aglycone with hydroxyl group in C-3
	[Agl + H-2H2O]+	Aglycone with a second hydroxyl group
ESI (−)	[M – 162-H]− strong peak	Saponin with Glc at C-28	LC-ESI-MS/MS LC-ESI-MS/MS
	[M – 162-H-162-172]−	Saponin with Glc at C-28, # of hexoses at C-3 and 172 corresponds to acyl moiety (Acyl-H2O)
APCI (−)	[Agl-H]− strong peak	Aglycone	LC-APCI-MS/MS

^aAgl (Aglycone); ESI (Electrospray); APCI (Atmospheric Pressure Chemical Ionization).

In conclusion, there are different techniques to analyze the quinoa SAPs by LC-MS/MS which used different ionization methods, ESI and APCI in the negative and positive mode being the most common ionization techniques. ESI and APCI in the positive mode provide similar information, while in the negative mode APCI gives information of sapogenin which is not observed in ESI. Thus, it is clear that no one technique will provide all of the desired identifying information. Consequently, simultaneous recording of the APCI in positive and negative modes is typically employed to get more information. In addition, it is common to combine GC-MS and LC-MS/MS to have more information for the identification, because the sapogenin could be identified by GC-MS, and the identification could be completed by LC-MS/MS, which is really important for the MS/MS analysis of some selected ions to get the sequence of saccharides in C-3. Finally, it is usually common to do a previous acid hydrolysis and basic hydrolysis to analyze the quinoa SAPs by GC-MS and LC-MS/MS to get more information about the structure, but in general it is not possible to determine the exact structure, because the MS does not give the stereochemical information, and some fragments, like those corresponding to hexoses and pentoses, could correspond to different molecules. Then, the LC-MS/MS gives a lot of information about the structure of quinoa SAPs but not the exact structure.

4. Quantitative Analysis

4.1. Direct Quantification of Saponins

4.1.1. Standards

The selection of appropriate standards for quantitative analysis of quinoa saponins (SAPs) has been shown to be a challenging task, due the complex mixture of SAPs in C. quinoa Madl et al. (2006) reported up to 87 different quinoa SAPs using LC-MS/MS, highlighting the difficulty of isolating individual compounds for use as standards. Moreover, no commercial quinoa-specific SAP standards are currently available, and then all reports about the quinoa SAPs quantification were done using a purified fraction of quinoa SAPs as standard or similar compounds from other sources.

In practice, a purified mixture of quinoa SAPs has been commonly employed as standards for total SAPs quantification in quinoa grains. ,,,,,− However, strict quality control is required to ensure the absence of coextracted glycosylated phenolics and simple sugars. Also, pure commercial SAPs and similar compounds have been used as standards for quinoa SAPs quantification, like saponin A, saponin B, saponin C, alfa-hederin, saponin Quillaja sp, hederacoside C, ,, and soyasaponin I. , Additionally, pure sapogenins such as oleanolic acid (OA) ,,− and steroidal compounds like diosgenin have been used as a standard for total sapogenin quantification, which is directly related to SAPS quantification.

In the analysis of quinoa SAPS, internal standards are employed to improve the accuracy and reproducibility of quantification. In GC–MS analyses, cholesterol decanoate has been used due to its chromatographic behavior being comparable to that of sapogenins. On the other hand, LC–qToF–MS/MS methods frequently employ hederacoside C and α-hederin (both at 2 μM) as structural analogues of quinoa SAPs, enabling more precision.

4.1.2. Afrosymmetric

The afrosymmetric methods are based on the surfactant properties of SAPs, and the height of stable and persistent foam is measured manually after the grain has been shaken in covered tubes containing water. They provide a rapid and economical estimate of total grain SAP content. , The first standardized quantification was presented by Koziol, even though there are previous reports using this kind of method. This method has been broadly accepted, and it is widely used due its rapid, cheap, and practical implementation, and despite its simplicity, the method has an acceptable detection limit (i.e., 0.01%). Unfortunately, this method is at best, semiquantitative because there is a tendency for a wrong estimation of SAP content because the quinoa has other surfactants, and some bidesmosdic SAPs could not form a stable foam. Despite the limitations mentioned, this method has been useful, given the challenging nature of SAP analysis. The high polarity and structural complexity of these compounds bring difficulties in their isolation, identification, and analytical quantification.

Several modified methods have been developed; the most used methods are the standard afrosymmetric method and the fast afrosymmetric method. Their main difference is the assay time, 73 and 7 min, respectively.

Recently, a novel microtiter macro lens-coupled smartphone (MCS) assay has been developed and validated for the quantitation of the total SAPs in quinoa, based on foam measurement with accessible technology and analyzed by software specially developed.

4.1.3. Thin Layer Chromatography

Thin layer chromatography (TLC) has been used to separate, quantify, and detect SAPs from different plant sources. Both phases, normal and reversed, have been used in several techniques such as TLC, high performance thin layer chromatography (HPTLC), and two-dimensional thin layer chromatography (2D-TLC), offering outstanding qualitative insights. When coupled with a computer featuring a dual-wavelength flying-spot scanner and two-dimensional analytical software, this method becomes suitable for the routine determination of SAPs in plant material. As a standard procedure, SAPs are identified on TLC by applying a solution of 10% H₂SO₄ in ethanol, Liebermann-Burchard reagent, or a mixture of p-anisaldehyde, sulfuric acid, and acetic acid, and under these conditions, triterpene SAPs exhibit blue-violet spots upon heating.

A specific TLC quantitative method is reported for C. quinoa. This method allows analyzing both the total SAP content and the composition in different quinoa plant tissues. The SAP composition has been determined according to the three main saponin groups found in quinoa, which contain OA (1), HD (2), and PA (3), as aglycone in each group. The visualization staining mixture was p-anisaldehyde, sulfuric acid, and glacial acetic acid, which produced a blue violet spot. The plate was then scanned using a TLC scanner configured to monitor at 575 nm.

4.1.4. UV–vis Spectroscopy

There are no UV–vis methods available to directly detect and quantify SAPs from C. quinoa; all of them must be carried out through colorimetric assays.

A method usually used is the presented by Hiai et al., which uses a sulfuric-vanillin solution to quantify triterpenoid SAPs at a wavelength of 544 nm and can be used to quantify SAPs as well as sapogenins. Even though, this method is not specific for SAPs and can generate color with other metabolites, variations have been developed to quantify triterpenes, , phenolic compounds, volatile terpenes, , and some sugars as rhamnose (at 470 nm) and sorbose (520 nm). This reaction was applied for the direct quantification of quinoa SAPs at 538 nm, but some modified methods have been reported, as the replacement of the sulfuric acid by a mixture of acetic acid-perchloric acid and measuring between 545 and 550 nm. ,,,,

Another spectrophotometric method commonly used and initially developed by Baccou and his collaborators uses p-anisaldehyde in sulfuric acid to specifically reveal sapogenins. This method has been adapted in several studies to directly quantify SAPs in quinoa. The method was later improved to be enough specifically to directly quantify SAPs in a complex analytical matrix without performing prior treatment, ensuring a reliable total quantification in complex extracts with no interference at 600 nm.

Finally, possibly the most widespread method is the Liebermann-Burchard test, which is not only applied qualitatively, but quantitatively. It is a test for unsaturated steroids (e.g., cholesterol) and triterpenes based on the formation of a blue-green color produced by the reaction of the sterol with acetic anhydride in the presence of concentrated sulfuric acid as shown in Figure . This test has been applied on whole SAPs at 528 or 580 nm. ,,,− A comparative study between the UV–vis Liebermann-Burchard based method and the afrosymmetric method shows that it is efficient for use in industry as it is precise, simple, and rapid to complete the analysis. Nevertheless, the foam method could be suitable for unwashed quinoa, given its quicker execution, absence of harmful chemicals, and the sufficiently large stable foam height that allows relatively accurate measurements.

12.

Reaction and products from the Liebermann–Burchard reaction for cholesterol (I _max maximum intensity).

Additionally, a modified and simplified Liebermann-Burchard method was proposed and used to a lesser extent. ,, The main difference is that the Liebermann-Burchard mixture was simplified to a mixture sulfuric acid/acetic acid, and the absorbance is measured at 527 nm, , which requires less care to prepare but can be less selective than the original. Even this method was adapted to use glycolic acid instead of acetic acid.

4.1.5. Quantification by HPLC and LC-MS

As we mentioned in the previous paragraphs, different analytical methods have been developed to quantify quinoa SAPs, such as afrosymmetric, hemolytic, spectrophotometric, and gas chromatography among others. However, the most common reported methods use high-performance liquid chromatography (HPLC) coupled to diode array detection (DAD) and liquid chromatography (LC) coupled to MS. The HPLC method is suitable for the direct analysis of SAPs in crude extracts; one of its advantages over other methods is its high accuracy and precision. However, there is the need for suitable SAP standards (see section 4.1.1) for the correct identification of the components and their quantitative determination. In this sense, most methods are aimed at the direct quantification of SAPs as analytes, generally using saponin C or soyasaponin I as standards. ,,,, On the others, the standard of SAPs for HPLC quantification can be obtained by purification of SAPs from crude extract using Sephadex LH-20 and other chromatographic techniques. ,

The HPLC coupled to UV/DAD showed high linearity with respect to SAPs from 210 to 220 nm; in addition, HPLC with a DAD detector can provide 3D fingerprint spectra which helps us to understand the effectiveness and accuracy results about the analyte. However, there are some inconveniences for SAPs in HPLC-UV/DAD analysis because SAPs does not have a chromophore group which is necessary for UV–vis detection. Then the detection at a low wavelength can limit the selection of solvents in the mobile phase; for example, the use of acetonitrile is better than methanol because acetonitrile absorbs lower than methanol. This is the reason why almost all the methods use a combination of water–acetonitrile, and the peak areas can be influenced by changes in the wavelength around 210 nm.

However, there are some considerations to take into account prior to HPLC injection: the system back pressure of the mobile phase, the viscosity, and temperature, analyzing these parameters can help us to avoid overlapping signals. The following Table shows some LC-conditions and detectors used in the analysis of quinoa SAPs by several authors.

8. Some Conditions Used in the HPLC Analysis of Quinoa SAPs .

	sample extraction			LC conditions
sample matrix	solvent	time (h)	method	column	gradient/mobile phase	flow rate (mL/min)	detection	ref
Raw, brand, quinoa	Methanol	24	Soxhlet	4 mm × 250 mm packed with LiChrospher 100 CH-8/2	25 to 40% acetonitrile in water by 15 min	2	LC-UV (200 nm)
Seeds	Hydro ethanolic solution	NR	Magnetic stirring	Gemini-NX RP C18 (Phenomenex, 250 × 4.6 mm i.d.; 5 μm particle size)	0.1% formic acid solution (A) and acetonitrile (B)	1.2	LC-DAD (209, 210, 211 nm)
Grains	Methanol/water/acetic acid	NR	Ultrasonic extraction	Kinetex C18 column (100 mm 4.6 mm, 2.6 μm)	(A) 1% acetic acid in water	0.8	LC-DAD (240, 280, 330 nm)
					(B) 60% phase A and 40% ACN
Seeds	Methanol followed by n-butanol	NR	Direct sonication	ACE 3 C18-AR column (150 mm × 4.6 mm, 3 μm particle size)	(A) 0.05% TFA	0.4	LC-DAD 210 nm
					(B) ACN with 0.05% TFA

^aNR = not reported.

4.1.6. Other Methods

SAPs have hemolytic activity. The hemolytic activity of SAP has been attributed by some researchers to the chemical interaction of saponin with cholesterol in the cell membrane. Not all hemolytic SAPs are from cholesterol, this is especially true for acid SAPs, as in C quinoa. Complete hemolysis occurred at concentrations equal to or lower than those necessary to form a single monolayer on the cell surface. The method for quinoa is an adaptation of an original method developed for alfalfa. , The hemolytic assay has been used to study the effect of extraction methods and chemical modifications of quinoa’s SAPs on the hemolytic and related biological activities, ,, as well as their quantification. The hemolytic activity can be measured by a colorimetric assay targeting the heme concentration in solution under wavelengths of 415 nm (microplate reader), at 540 nm (UV–vis). , Another method used to measure hemolysis is by erythrocyte sedimentation. It is important to consider that monodesmosidic SAPs have an increased hemolytic activity compared to bidesmosidic SAPs. It can be attributed to the unique detergent-like properties of monodesmosides, which enable them to selectively lyse biomembranes.

The WDST (water droplet surface tension) method is a nontraditional and innovative alternative. The method has been used for the measurement of surfactants in the mining and oil extraction industries, but it was adapted to quantify SAPs from quinoa due to their surfactant qualities.

Infrared spectra showed characteristic signals indicating the presence of compounds with chemical groups such as alcohol (3400–3200 cm^–1), alcohol or amino groups (3019 cm^–1), carboxyl (1725 cm^–1), and esters (1730 cm^–1), corresponding to the chemical structure reported for SAPs. The IR results for the composition of the extracts after 24 and 72 h showed some differences in relation to the transmittance of the bands and differences in the signals.

4.2. Quantification of Sapogenins

4.2.1. Standards

The most common standards used for sapogenins quantification are HD (2), PA (3), and OA (1), ,,, which are also the main sapogenins present in C. quinoa. Other studies used single phytosteroids as diosgenin or, less common standards as heptadecane, margarinic acid, and cholesterol.

4.2.2. Hydrolysis of Saponins

The procedure for converting SAPs to sapogenins involves the hydrolysis of the ester and/or ether bonds in SAPs. In this context, SAPs can undergo hydrolysis through chemical means (using acid or alkali), enzymatic reactions, and microbial methods. This results in the generation of sapogenins, pro-sapogenins, sugar residues, or monosaccharides, depending on the specific hydrolysis method and conditions applied. The acid hydrolysis of quinoa SAPs is a useful procedure to isolate and identify the respective sapogenins or their conjugated sugars. ,, Several analytical methods include an acid hydrolysis step to quantify quinoa sapogenins by different instrumental methods like GC-MS ,,,, and LC-MS. A disadvantage of the chemical hydrolysis, i.e. mineral acids, is the degradation of the produced sapogenins during the hydrolysis reaction. To ensure the highest sapogenin levels, it is recommended to limit the hydrolysis time to a maximum of 1–3 h. Extended acid hydrolysis times may result in reduced sapogenin yields by degradation.

4.2.3. UV–vis Spectroscopy

The methods used to quantify quinoa sapogenins are practically the same as those used for the quantification of SAPs. However, the main differences are the recording wavelength and selection of standards.

The colorimetric method based on the sulfuric-vanillin reaction, was used to quantify quinoa SAPs directly (see Section 4.1.4), but it can also be applied to quantify sapogenins at 460 nm.

Another colorimetric method to quantify sapogenins, that is also useful to quantify SAPs directly, uses p-anisaldehyde in sulfuric acid. Various standards of steroidal and triterpenoid sapogenins were tested, and triterpenoid sapogenins had a maximum absorbance close to 540 nm, while for steroid sapogenins, the maximum absorbance was around 460 nm with different molar absorption coefficients. Furthermore, the amount of sulfuric acid represents more than 60% (v/v) in the reaction and causes the formation of interferences close to the quantification wavelengths; for example, rhamnose absorbs at 470 nm, and sorbose absorbs at 520 nm. These interferences may come from the plant matrix or from the glycosides present in the SAPs themselves, making it necessary to perform a pretreatment before performing the quantification.

4.2.4. GC-MS Method to Quantify Sapogenins

In previous paragraphs (see section 3.2), we described the methodology to identify quinoa SAPs by GC-MS, the same methodology can be used to quantify quinoa SAPs. The total SAPs and total sapogenins content are important to evaluate, and the GC-MS is a reliable tool in the analytical process. The reports using this method GC-MS about the structural properties of quinoa SAPs determined that the major sapogenins in quinoa are OA (1), HD (2) and PA (3). It has also been demonstrated that quinoa SAPs are found in all parts of the plant, the major concentration of SAPs are located in the episperm of the grain and the concentration ranges varies from 0.01% to 5% among the varieties.

The method is based on the hydrolysis of SAPs to their sapogenins, and their subsequent separation and derivatization as OA (1), HD (2), and PA (3) trimethylsilyl ethers (Figure ) which are quantified in diverse matrixes based on calibration curves of each sapogenin derived. In general, chromatographic procedures are applicable only to aglycones (sapogenols) derived from intact SAPs by acid or enzymatic hydrolysis. However, an estimate of the saponin content can be made from the combination of these quantitative chromatographic data on sapogenols and the qualitative information on the molecular weights of intact SAPs that is available from atomic bombardment mass spectral studies. On the other hand, total sapogenins can be estimated quantifying them with respect to one of the major sapogenins, such as OA (1), and then using a conversion factor between SAPs and sapogenins (% SAPs = 8.471 x% Sapogenins), which is found using the weight of the purified SAPs used for the hydrolyzation, and the determined total weight of sapogenins, the percentage of total SAPs can be obtained.

4.2.5. HPLC Methods to Quantify Sapogenins

The evaluation and identification of individual sapogenins on a hydrolyzed extract were analyzed by GC-MS which involves many steps prior to injection such as sapogenin derivatization. But the HPLC technique offers direct quantification of sapogenins, showing speed analysis, high resolution, and high sensitivity. However, some factors could affect the analysis by HPLC such as the polarity of mobile phase composition, temperature, flow rate of the mobile phase, column temperature, column types, and detectors (Table ).

According of Lozano et al. (2013), an analysis by HPLC revealed the presence of four major sapogenins in quinoa sapogenins extract; the analysis were performed on an HPLC system (Agilent, 1100 series) equipped with a quaternary pump and DAD detector, and the separation was done in an Eclipse Plus C 18 column (125 × 4.6, 5 μm) by isocratic elution with formic acid (0.1%) (A) and methanol (B) with a ratio of 15:85 (A:B, v/v) at room temperature with a flow rate of 1.0 mL/min. All of the sapogenins were detected at 210 nm. The result of separation, identification, and quantification shows OA (1) (13.3 min, 24%), SA (4) (6.2 min, 12%), HD (2) (5.4 min, 28%), and PA (3) (2.8 min, 27%). Additionally, Carpio-Paucar. et al. (2023) reported the determination of OA (1) and HD (2) in white and red quinoa extracts using methanol:acid water (89:11) as a mobile phase solution, and the separation was performed in a C18 Reversed Phase HPLC Column, 5 μm, 2.1 mm × 20 mm, identifying all sapogenins by the DAD detector. The individual sapogenins quantified in white quinoa were OA (1) and HD (2) with 5.61 ± 0.02 and 9.43 ± 0.02 mg/L, respectively; red quinoa also showed the presence of OA (1) 5.30 ± 0.01 mg/L and HD (2) 14.44 ± 0.01 mg/L.

In general, the HPLC methods are more used to identify SAPs instead of sapogenins, and the concentration of the different aglycones could vary greatly among the different varieties of quinoa seeds, even though it is important to know that it is also possible to use this method to identify and quantify sapogenins.

4.2.6. Comparative Analysis of Quantitative Methods

This Account compares the main analytical methods reported for the quantification of quinoa saponins (SAPs) and sapogenins. The evaluated parameters include analytical range, linearity (curve equation and R ²), limit of detection (LOD), and limit of quantification (LOQ), as summarized in Table .

9. Analytical Parameters of Linearity and Sensitivity for Selected Methods .

method	range	equation	R 2	LOD	LOQ	ref
Direct SAP Analysis
Afrosymmetric (semiquantitative)	0–400 mg/mL	$$\begin{array}{l}y=7.638x+0.173\\ (\mathrm{Standard\; SAP\; mixture})\end{array}$$	0.992	0.20 mg/mL	0.60 mg/mL
Afrosymmetric rapid (semiquantitative)	0–400 mg/mL	$$\begin{array}{l}y=0.668x+0.174\\ (\mathrm{Standard\; SAP\; mixture})\end{array}$$	0.935	NR	NR
Hemolytic assay (semiquantitative)	0.1–0.8%	$$\begin{array}{l}y=0.000009x^3+0.00121x^2+0.0551+0.00675\\ (\mathrm{Commercial\; SAP\; preparations})\end{array}$$	0.998	NR	NR
UV–vis (sulfuric vanillin)	3.08–24.61 μg/mL	$$\begin{array}{l}y=0.072x+0.008\\ (\beta -\mathrm{sitosterol})\end{array}$$	0.9998	0.042 μg/mL	0.14 μg/mL
UV–vis (p-anisaldehyde)	19–230 μg/mL	$$\begin{array}{l}y=5.95\times {10}^{-3}x+22.5\times {10}^{-3}\\ (\mathrm{Escin\; IB})\end{array}$$	0.9934	6 μg/mL	19 μg/mL
	18–230 μg/mL	$$\begin{array}{l}y=7.30\times {10}^{-3}x+46.1\times {10}^{-3}\\ (\mathrm{Protodioscin})\end{array}$$	0.9918	5 μg/mL	18 μg/mL
	20–230 μg/mL	$$\begin{array}{l}y=5.08\times {10}^{-3}x+55.4\times {10}^{-3}\\ (\mathrm{Escin})\end{array}$$	0.9973	6 μg/mL	20 μg/mL
UV–vis (Liebermann Burchard)	0.05–0.65 mg/mL	$$\begin{array}{l}y=4.5725x+0.0164\\ (\mathrm{oleanolic\; acid})\end{array}$$	0.9998	0.05 mg/mL	0.05 mg/mL
HPLC-DAD ESI-TOF-MS	0.577–1000 μg/mL	$$\begin{array}{l}\mathrm{y}=143.9\mathrm{x}+32078.0\\ (\mathrm{oleanolic\; acid})\end{array}$$	0.9967	0.243 μg/mL	0.577 μg/mL
HPLC-DAD	32–256 μg/mL	$$\begin{array}{l}\mathit{y}=4442.3\mathit{x}-14.73\\ \mathrm{alfa}-\mathrm{hederin}\end{array}$$	0.9999	1.24 μg/mL	4.15 μg/mL
HPLC-DAD	NR	$$\begin{array}{l}\mathit{y}=3262446.4808\mathit{x}+1091043.3256\\ (\mathrm{Sapinin\; C})\end{array}$$	0.9899	NR	NR
WDST (Semiquantitative)	0.0–1.6 mg/mL	$$\begin{array}{l}{x}_0=e+\log (\frac{d-c}{Y_0-c}-1)/b\\ (\mathrm{Saponin\; standard}(\mathrm{Quillaja\; saponaria},\mathrm{Molina}))\end{array}$$	NR	NR	NR
Sapogenines Analysis
UV–vis (sulfuric-vanillin)	NR	NR	NR	NR	NR
UV–vis (p-anisaldehyde)	0–25 nmol	$$\begin{array}{l}y=49151\mathit{x}\\ (\mathrm{Diosgenin},\mathrm{Tigogenin},\mathrm{Smilagenin},\mathrm{Hecogenin})\end{array}$$	NR	0.5 μg/mL	0.5 μg/mL
GC-MS	0.01–1 mg/mL	NR (Oleanolic acid, Hederagenine, Phytolacagenic acid, Serjanic acid)	0.98	NR	NR
GC-MS/MS	NR	$$\begin{array}{l}y=3.9900\mathit{x}-0.2097\\ (\mathrm{oleanolic\; acid})\end{array}$$	0.993	0.003 mg/mL	0.007 mg/mL
		$$\begin{array}{l}y=3.4719\mathit{x}-0.1708\\ (\mathrm{Hederagenin})\end{array}$$	0.994	0.003 mg/mL	0.008 mg/mL
		$$\begin{array}{l}y=0.8079\mathit{x}-0.0468\\ (\mathrm{Phytolaccagenic\; acid})\end{array}$$	0.998	0.013 mg/mL	0.028 mg/mL

^aNR = Not reported.

^bThe Standard used in the calibration curve is specified in parentheses () in the equation column.

^cCalculated using the σ = value used in the reference. $\mathrm{L}\mathrm{O}\mathrm{D}=3.3\times \frac{\sigma }{S}$ ; $\mathrm{L}\mathrm{O}\mathrm{Q}=10\times \frac{\sigma }{S}$ .

For saponins, the widely used afrosymmetric method offers simplicity and low cost but presents important drawbacks such as a narrow analytical range, poor linearity, and low sensitivity. Moreover, some studies reported weak correlation with others, while in others, standards were not employed, further limiting reliability. In contrast, the UV–Vis vanillin–sulfuric method displayed high sensitivity and excellent linearity, whereas the Liebermann–Burchard assay achieved good linearity but comparatively lower sensitivity. Among instrumental chromatographic approaches, HPLC-DADparticularly the method described by Verza et al. (2017).showed outstanding linearity and sensitivity.

Regarding sapogenins, few direct detection methods have been reported; however, GC-MS and GC-MS/MS, such as the protocol described by Jeong Gyu Lim et al. (2020), are widely applied and exhibit strong analytical parameters. Nevertheless, direct methods for SAP determination generally provide superior analytical parameters and may represent a more straightforward and promising strategy for future applications.

5. Conclusions

The quinoa SAPs play a significant role at the agro-industrial level due to their diverse applications. On one hand, they can be utilized directly in the development of various products, including pharmaceuticals, cosmetics, and natural pesticides, thanks to their bioactive properties. On the other hand, their removal is essential in food processing, mainly for their naturally bitter taste that can affect the sensory quality of quinoa-based food products. Therefore, understanding and managing quinoa SAPs content are crucial for optimizing both the industrial use and consumer acceptance of quinoa-derived products.

The quinoa SAPs are present as a complex mixture that exhibits structural and, particularly, stereochemical complexity. This complexity makes the isolation and structural identification of individual compounds a long, tedious, and expensive process requiring significant expertise and advanced methodologies. Their complex molecular structures consist of diverse glycoside compounds, varying aglycones, and multiple sugar moieties with diverse stereoisomeric forms, demanding the integration of various spectroscopic and chromatographic techniques for a proper characterization, such as NMR, LC-MS/MS, and GC-MS to accurately determine their composition and configuration. In some cases, their structural complexity is so high that partial hydrolysis of the SAPs is necessary to break down glycosidic bonds and facilitate the identification of individual aglycones and sugar units. These challenges highlight the need for continuous advancements in analytical methodologies to improve the efficiency and accuracy of quinoa SAP characterization. The future trends in analytical techniques and new generation equipment, like LC-NMR, LC-MS/MS, and high-resolution NMR (1.2 GHz), using machine learning as a tool, could help to accurately identify quinoa SAPs in bioactive fractions, avoiding the long and expensive process of isolation. Until now, approximately 90 SAPs were determined in quinoa extracts, but only about 30 were isolated and identified using NMR. The remaining SAPs were detected through GC-MS and LC-MS/MS techniques, which do not allow for exact structural elucidation.

This complexity also presents important challenges for SAPs quantification, since obtaining a pure standard applicable to all cases is extremely difficult because each variety of quinoa can contain a unique profile of SAP, with different concentrations and compositions, which complicates standardization efforts. Furthermore, quinoa SAPs absorb around 210 nm, where several other organic compounds absorb, making it essential to use and develop advanced analytical techniques and standardized methodologies to ensure accurate quantification in different quinoa varieties.

In general, food industry applications do not require the precise identification or quantification of individual quinoa SAPs. Instead, the main interest lies in determining the total SAPs content of a product as this serves as a critical parameter for quality control, product standardization, and regulatory compliance. For this purpose, industries typically rely on fast, cost-effective, and scalable analytical methodssuch as UV–vis and afrosymmetric assaysbecause of their simplicity and ability to provide rapid estimates of SAPs levels. Among these, UV–vis methods have shown superior sensitivity and linearity. In contrast, the cosmetic and pharmaceutical industries demand more advanced analytical approaches, such as LC-MS/MS or GC-MS, which enable a more detailed characterization of the bioactive quinoa SAPs present in products.

The need for these practical approaches arises from the high demand for quinoa and its derivatives in food, pharmaceutical, and cosmetic industries, where quinoa SAPs content directly impacts the product quality, sensory attributes, and consumer acceptance. As a result, although advanced analytical techniques are essential for research and detailed characterization, some companies prioritize methods that balance accuracy, speed, and cost-effectiveness to meet production and market demands efficiently.

Glossary

Abreviations

A549

Cell line of the lung adenocarcinoma

APCI

Atmospheric pressure chemical ionization

BSTFA

Bis (trimethylsilyl)­trifluoroacetamide

CC

Column chromatography

DAD

Diode array detector

D/CI

Desorption chemical ionization mass spectrometry

ESI

Electrospray ionization

FAB-MS

Fast atom bombardment mass spectrometry

FD

Field desorption mass spectrometry

GC-MS

Gas chromatography–mass spectrometry

HD

Hederagenin

HDL

High-density lipoprotein

HeLa

Cell line of human cervical adenocarcinoma

HepG2

Cell line hepatoma

HPTLC

High-performance thin layer chromatography

IC50

Half maximal inhibitory concentration

IL-6

Interleukin-6

ITMS

Ion-trap mass spectrometry

LC-MS

Liquid chromatograph–mass spectrometry

LC-MS/MS

Liquid chromatography–tandem mass spectrometry

LDL

Low-density lipoprotein

MALDI-MS

Matrix-assisted laser-desorption ionization

MTT

Methylthialazole tetrazolium

NMR

Nuclear magnetic resonance

NO

Nitric oxide

OA

Oleanolic acid

PA

Phytolacagenic acid

SA

Serjanic acid

SAP

Saponin

SH-SY5Y

Cell line of neuroblastoma

TFA

Trifluoroacetic acid

TLC

Thin-layer chromatography

TMS

Trimethylsilyl ethers

TMSi

1-(Trimethylsilyl)­imidazole

TNF-α

Tumor necrosis factor alpha

TOF-MS

Time-of-flight-mass spectrometry

UV

Ultraviolet

μM

Micromolar

R.V. and G.R.A. contributed to the preparation, editing, and review of all the information provided in this manuscript. In addition: G.R.A.: review and compilation of all the general aspects and properties of quinoa saponins. R.V.: review and compilation of the quantitative analytic methods reported for quinoa saponins. Y.R.F. and M.L.: review and compilation of the NMR data of quinoa saponins. M.L. and G.R.A.: review and compilation of the MS data of quinoa saponins. Y.K. and Y.F.: review and complementation of quinoa saponins generalities

The authors declare no competing financial interest.