Free amino acids in African indigenous vegetables: Analysis with improved hydrophilic interaction ultra-high performance liquid chromatography tandem mass spectrometry and interactive machine learning.

A hydrophilic interaction (HILIC) ultra-high performance liquid chromatography (UHPLC) with triple quadrupole tandem mass spectrometry (MS/MS) method was developed and validated for the quantification of 21 free amino acids (AAs). Compared to published reports, our method renders collectively improved sensitivity with lower limit of quantification (LLOQ) at 0.5~42.19 ng/mL with 0.3 µL injection volume (or equivalently 0.15~12.6 pg injected on column), robust linear range from LLOQ up to 3521~5720 ng/mL (or 1056 ~ 1716 pg on column) and a high throughput with total time of 6 min per sample, as well as easier experimental setup, less maintenance and higher adaptation flexibility. Ammonium formate in the mobile phase, though commonly used in HILIC, was found unnecessary in our experimental setup, and its removal from mobile phase was key for significant improvement in sensitivity (4~74 times higher than with 5 mM ammonium formate). Addition of 10 (or up to100 mM) hydrochloric acid (HCl) in the sample diluent was crucial to keep response linearity for basic amino acids of histidine, lysine and arginine. Different HCl concentration (10~100 mM) in sample diluent also excreted an effect on detection sensitivity, and it is of importance to keep the final prepared sample and calibrators in the same HCl level. Leucine and isoleucine were distinguished using different transitions. Validated at seven concentration levels, accuracy was bound within 75~125%, matrix effect generally within 90~110%, and precision error mostly below 2.5%. Using this newly developed method, the free amino acids were then quantified in a total of 544 African indigenous vegetables (AIVs) samples from African nightshades (AN), Ethiopian mustards (EM), amaranths (AM) and spider plants (SP), comprising a total of 8 identified species and 43 accessions, cultivated and harvested in USA, Kenya and Tanzania over several years, 2013~2018. The AN, EM, AM and SP were distinguished based on free AAs profile using machine learning methods (ML) including principle component analysis, discriminant analysis, naïve Bayes, elastic net-regularized logistic regression, random forest and support vector machine, with prediction accuracy achieved at ca. 83~97% on the test set (train/test ratio at 7/3). An interactive ML platform was constructed using R Shiny at https://boyuan.shinyapps.io/AIV_Classifier/ for modeling train-test simulation and category prediction of unknown AIV sample(s). This new method presents a robust and rapid approach to quantifying free amino acids in plants for use in evaluating plants, biofortification, botanical authentication, safety, adulteration and with applications to nutrition, health and food product development.

Rapid screening of glycoalkaloids in Solanum scabrum and S. nigrum berries using ultra-high-performance liquid chromatography with pathway-specified in-source fragmentation tandem mass spectrometry.

RATIONALE: The safe consumption of Solanum scabrum and S. nigrum berries (SNBs) depends on a reliable and rapid chemical screen for the testing of the fruit and/or final food and industrial products for the presence and level of toxic glycoalkaloids. Such a rapid and sensitive screen could also be used by those involved in food safety and forensics, industry, research labs and those in agriculture production, breeding and food processing. Significant variation in the content and composition of glycoalkaloids across SNBs has been reported. To facilitate high-throughput targeted analysis, this work overcame the slow scan speed of a traditional triple quadruple mass spectrometry (QqQ) method by development of a pseudo-MS3 method. METHODS: In-source fragmentation functioned as a pseudo-MS or pseudo-hydrolysis to trim down the structurally diverse and complex glycosides into five types of aglycone ions, which were then analyzed using multiple reaction monitoring (MRM). Characteristic product ions were selected based on the aglycone skeleton and substitution pattern and associated fragmentation pathway. RESULTS: A compact method with only 15 MRM transitions were developed for high-throughput screening of very diverse glycoalkaloids. Glycosides of the same aglycone type were readily identified in the same transition window without the need for mass spectra interpretation. Validated using solamargine, the sole available standard, the accuracy was 99.7-101.3%, the intra- and inter-day precision were, respectively, 2.5-5.0% and 8.0-9.2%, and the lower limit of detection and quantification were, respectively, 3.1 and 10.2 ng/mL (with 1 µL injection volume). CONCLUSIONS: The peudo-MS3 method allowed for high-throughput targeted analysis with compact MRM transitions to address a large number of glycoalkaloids with diverse structures. This method could serve to meet the most heavy-duty demand for rapid inspection of glycoalkaloids in SNBs. This method can be adopted and used by those involved in food safety and forensics, in developing food and industrial products and in genetics and breeding.

Simultaneous quantification of polyphenols, glycoalkaloids and saponins in African nightshade leaves using ultra-high performance liquid chromatography tandem mass spectrometry with acid assisted hydrolysis and multivariate analysis.

This study developed comprehensive quantification methods for major nutritive and antinutritive phytochemical aglycones in edible African nightshade leaves, an underutilized food resource in the sub-Saharan area. A simultaneous hydrolysis and extraction method was developed using methanol with 2 M sulfuric acid with incubation at 65 °C for 60 min. UHPLC-QqQ-MS/MS methods were developed and validated for hydrolysis optimization and for quantification of eight major aglycones of polyphenols, alkaloids and sapogenins in 20 differently sourced nightshade leaves, comprising two African species Solanum scabrum and S. nigrum, and from two distinct cultivation sites, one in New Jersey, US and the other in Kenya Eldoret. Variation in species, accessions and cultivation environment played an important role in affecting the phytochemical profile. Total antinutritive alkaloids and sapogenins in all nightshade leaves were evaluated and found to be safe for consumption. This work provides evidence that the consumption of African nightshade leaves as a nutrient rich leafy green vegetable is safe and can contribute to food security and nutritional improvement in the sub-Saharan area.

Identification of Polyphenols, Glycoalkaloids, and Saponins in Solanum scabrum Berries Using HPLC-UV/Vis-MS.

Consumption safety of Solanum scabrum berries is controversial in different cultural practices and evaluation of the toxicity as well as micronutrition value relies on relevant phytochemical study. Thus, this study aimed to systematically profile the phytochemicals in the berries from different genetic sources and maturity. Using a combination of three different and complementary methods of HPLC-UV/Vis-MS or MS/MS with acid-assisted hydrolysis, a total of 54 phytochemicals were identified including polyphenols, saponins and toxic glycoalkaloids. Particularly, a broad range of glycoalkaloids of solasodine and its uncommon or potentially novel hydroxylated and methylated derivatives were reported, with the structure putatively identified based on the known scaffold-fragmentation pattern. Other identified phytochemicals included phenolic acids of chlorogenic acid and neochlorogenic acid, flavonol glycosides of quercetin and isorhamnetin, anthocyanins of petunidin, malvidin and delphinidin, and saponins of diosgenin and tigogenin. PRACTICAL APPLICATION: This study provides solutions for identifying the phytochemicals of S. scabrum berries, and unveiled for the first time a wide range of toxic glycoalkaloids of solasodine and analogues in the berries from different genetic sources and maturation stages. This work laid the foundation for prospective quantitative determination of berry phytochemicals and future toxicity and nutrition evaluation, and could also apply to facilitate screening or breeding for glycoalkaloid-deficient genotypes that can be used as new food supply.

Quantity assessment of polyphenols, glycoalkaloids and saponins in Solanum scabrum berries of different genetic sources and maturity by HPLC/UV-visible/MS methods.

BACKGROUND: Solanum scabrum berries in sub-Saharan Africa are prolific but neglected as an agricultural resource. Recognition and application of such underutilized resources rely on systematic study of the relevant phytochemicals of commercial value. RESULTS: The quantities of a total of 54 phytochemicals in Solanum scabrum berries were assessed using HPLC-MS methods. Berries from eight different genetic sources were analyzed with two entries monitored across different maturation stages. There was a significant variation among mature berries in the accumulation of phenolic acids, 91.5-794 mg·100 g-1 dry weight (DW); flavonols, 76.3-897 mg·100 g-1 DW; anthocyanins, 178-4650 mg·100 g-1 DW; glycoalkaloids, 1.76-1630 mg·100 g-1 DW; and saponins, 82.2-606 mg·100 g-1 DW. Fruit development from immature to post-frost harvest featured dynamic changes in phytochemical composition and, despite remarkable differences in the absolute magnitude of content, the trend of change was generally similar in different genetic sources. CONCLUSIONS: The genotype-dependent difference in toxic glycoalkaloids in mature berries may partially explain the consumption controversy as it reflects glycoalkaloid content. The analytical methods applied in this work should serve for quality control of glycoalkaloids thereby improving the safe utilization of this berry. In addition, the selection and breeding of new genotypes with low and safe levels of glycoalkaloids and saponins in the berry could be of value in sub-Saharan Africa to increase nutrition and generate new income opportunities for growers. © 2019 Society of Chemical Industry.