A High-Throughput Extraction and Analysis Method for Steroidal Glycoalkaloids in Tomato

Abstract

Tomato steroidal glycoalkaloids (tSGAs) are a class of cholesterol-derived metabolites uniquely produced by the tomato clade. These compounds provide protection against biotic stress due to their fungicidal and insecticidal properties. Although commonly reported as being anti-nutritional, both in vitro as well as pre-clinical animal studies have indicated that some tSGAs may have a beneficial impact on human health. However, the paucity of quantitative extraction and analysis methods presents a major obstacle for determining the biological and nutritional functions of tSGAs. To address this problem, we developed and validated the first comprehensive extraction and ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) quantification method for tSGAs. Our extraction method allows for up to 16 samples to be extracted simultaneously in 20 min with 93.0 ± 6.8 and 100.8 ± 13.1% recovery rates for tomatidine and alpha-tomatine, respectively. Our UHPLC-MS/MS method was able to chromatographically separate analytes derived from 18 tSGA peaks representing 9 different tSGA masses, as well as two internal standards, in 13 min. Tomato steroidal glycoalkaloids that did not have available standards were annotated using high resolution mass spectrometry as well as product ion scans that provided fragmentation data. Lastly, we utilized our method to survey a variety of commonly consumed tomato-based products. Total tSGA concentrations ranged from 0.2 to 3.4 mg/serving and represent some of the first reported tSGA concentrations in tomatobased products. Our validation studies indicate that our method is sensitive, robust, and able to be used for a variety of applications where concentrations of biologically relevant tSGAs need to be quantified.

Genomic Selection in an Outcrossing Autotetraploid Fruit Crop: Lessons From Blueberry Breeding

“…Genomic information can also provide new opportunities to integrate biotechnology and quantitative genetics into modern breeding programs, creating platforms for both deliveries of new products and biological discovery (Hickey et al, 2017 ). For example, in blueberry, biological discoveries have been addressed via QTL mapping (Cappai et al, 2020a ) and GWAS studies (Ferrão et al, 2018 , 2020 ) for multiple fruit quality traits. Unifying such discoveries with prediction is challenging, but it has been addressed under three different avenues: (i) use of GWAS discovered QTL as fixed effects on GS models; (ii) incorporating markers (or QTL) in MAS designs, and (iii) using genome-editing technology to speed up breeding.…”

“…While QTL analyses are usually performed using structured populations, GWAS increases the mapping resolution by using populations with low levels of linkage disequilibrium considering a deep history of recombination events. In blueberry, we recently detected candidate genomic regions and markers associated with different fruit quality traits (Ferrão et al, 2018 ) and flavor-related volatiles (Ferrão et al, 2020 ) via GWAS investigations; and we built a high-density linkage map and detected QTL associated to berry firmness (Cappai et al, 2020a ). In counterpart, GS aims to predict breeding values by using all genome-wide markers simultaneously (Meuwissen et al, 2001 ).…”

Steroidal alkaloid biosynthesis is coordinately regulated and differs among tomatoes in the red‐fruited clade

“…The purpose of our work is to describe the chemical diversity of tomato steroidal alkaloids through a detailed and quantitative analysis (Dzakovich et al, 2020) of cultivated, landrace, and wild, red-fruited relatives of tomato and identify quantitative trait loci (QTL) associated with these metabolites. Populations were selected based on known patterns of genomic variation and were cultivated in multiple locations and years to separate genetic and environmental effects.…”

“…Steroidal alkaloids were extracted and quantified, as previously described, using a rapid extraction and ultra-high performance liquid chromatography tandem mass spectrometry (Dzakovich et al, 2020). Dehydrotomatidine, tomatidine, dehydrotomatine, alpha-tomatine, hydroxytomatine, acetoxytomatine, dehydrolycoperoside F/G/dehydroesculeoside A, lycoperoside F/G/esculeoside A, esculeoside B, and total steroidal alkaloids (the sum of all previous analytes) were quantified.…”

“…Dehydrotomatidine, tomatidine, dehydrotomatine, alpha-tomatine, hydroxytomatine, acetoxytomatine, dehydrolycoperoside F/G/dehydroesculeoside A, lycoperoside F/G/esculeoside A, esculeoside B, and total steroidal alkaloids (the sum of all previous analytes) were quantified. Alkaloids that have the same mass (e.g., lycoperoside F/G/esculeoside A) were collated together if they could not be differentiated (Dzakovich et al, 2020). Acetonitrile, formic acid, isopropanol, methanol, and water were of liquid chromatography mass spectrometry grade (Fisher Scientific).…”