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Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS.

ABSTRACT: The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both buffer and human plasma. We compared recovery, coverage, repeatability, matrix effects, selectivity and orthogonality of all methods tested for non-lipid metabolome in combination with reversed-phased and mixed-mode liquid chromatography mass spectrometry analysis (LC-MS). Our results confirmed wide selectivity and excellent precision of solvent precipitations, but revealed their high susceptibility to matrix effects. The use of all seven methods showed high overlap and redundancy which resulted in metabolite coverage increases of 34-80% depending on LC-MS method employed as compared to the best single extraction protocol (methanol/ethanol precipitation) despite 7x increase in MS analysis time and sample consumption. The most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether. Our results help facilitate rational design and selection of sample preparation methods and internal standards for global metabolomics.

SUBMITTER: Sitnikov DG

PROVIDER: S-EPMC5175266 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS.

Sitnikov Dmitri G DG Monnin Cian S CS Vuckovic Dajana D

Scientific reports 20161221

The comparison of extraction methods for global metabolomics is usually executed in biofluids only and focuses on metabolite coverage and method repeatability. This limits our detailed understanding of extraction parameters such as recovery and matrix effects and prevents side-by-side comparison of different sample preparation strategies. To address this gap in knowledge, seven solvent-based and solid-phase extraction methods were systematically evaluated using standard analytes spiked into both ...[more]

PMID: 28000704

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Project description:Proper sample preparation protocols represent a critical step for liquid chromatography-mass spectrometry (LC-MS)-based proteomic study designs and influence the speed, performance and automation of high-throughput data acquisition. The main objective of this study was to compare two commercial solid-phase extraction (SPE)-based sample preparation protocols (comprising SOLAµTM HRP SPE spin plates from Thermo Fisher Scientific and ZIPTIP® C18 pipette tips from Merck Millipore) for analytical performance, reproducibility, and analysis speed. The house swine represents a promising animal model for studying human eye diseases including glaucoma and provides excellent requirements for the qualitative and quantitative MS-based comparison in terms of ocular proteomics. In total six technical replicates of two protein fractions [extracted with 0.1% dodecyl-ß-maltoside (DDM) or 1% trifluoroacetic acid (TFA)] of porcine retinal tissues were subjected to in-gel trypsin digestion and purified with both SPE-based workflows (N = 3) prior to LC-MS analysis. On average, 550 ± 70 proteins (1512 ± 199 peptides) and 305 ± 48 proteins (806 ± 144 peptides) were identified from DDM and TFA protein fractions, respectively, after ZIPTIP® C18 purification, and SOLAµTM workflow resulted in the detection of 513 ± 55 proteins (1347 ± 180 peptides) and 300 ± 33 proteins (722 ± 87 peptides), respectively (FDR < 1%). Venn diagram analysis revealed an average overlap of 65 ± 2% (DDM fraction) and 69 ± 4% (TFA fraction) in protein identifications between both SPE-based methods. Quantitative analysis of 25 glaucoma-related protein markers also showed no significant differences (P > 0.05) regarding protein recovery between both SPE methods. However, only glaucoma-associated marker MECP2 showed a significant (P = 0.02) higher abundance in ZIPTIP®-purified replicates in comparison to SOLAµTM-treated study samples. Nevertheless, this result was not confirmed in the verification experiment using in-gel trypsin digestion of recombinant MECP2 (P = 0.24). In conclusion, both SPE-based purification methods worked equally well in terms of analytical performance and reproducibility, whereas the analysis speed and the semi-automation of the SOLAµTM spin plates workflow is much more convenient in comparison to the ZIPTIP® C18 method.

Project description:BackgroundPyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with potential hepatic and carcinogenic diseases in humans and animals. The concern over PAs has increased as the consumption of herbal medicines has increased.ObjectiveThis study aimed to develop and validate a sensitive analytical method to determine 28 PAs in five herbal medicines using liquid chromatography (LC)-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS). Additionally, this study identified and quantified the amount of PAs in 10 samples of each herbal medicine.MethodsThe pretreatment in the proposed LC-MS/MS analysis comprised solvent extraction using 0.05M H2SO4 in 50% methanol and clean-up step using an mixed-mode cationic exchange (MCX)-solid-phase extraction (SPE) cartridge. The PA contents in herbal medicines were measured by using the developed method.ResultsThe proposed method had recoveries ranging from 72.5-123.7% for the Atractylodis Rhizoma Alba, 70.6-151.7% for Alba Chrysanthmi Flos, 80.6-130.9% for Leonuri Herba, 70.3-122.9% for Gastrodiae Rhizoma, and 67.1-106.9% for Glycyrrhizae Radix. Even though a few samples showed recoveries in unsatisfactory values, the proposed method indicated entirely sufficient recoveries and precision in most samples. In monitoring results, only Leonuri Herba contained two PAs, which indicated Retrorsine (4/10) of 84.7-120.9 μg/kg and Senkirkine (10/10) of 60.9-170.7 μg/kg.ConclusionThe results obtained from this study demonstrate that the proposed method is fit for purpose to determine 28 PAs in herbal medicines. Therefore it could serve as a regulatory method capable of being used for controlling the risks of PAs in certain medicinal plants and dietary supplements.HighlightsAn LC-MS/MS method for the determination of 28 pyrrolizidine alkaloids in herbal medicines was developed and validated through this study. The proposed method is considered as an useful method for monitoring pyroolizidine alkaloids in herbal medicines.

Dataset Information

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS.

Publications

Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS.

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