Objective Set of Criteria for Optimization of Sample Preparation Procedures for Ultra-High Throughput Untargeted Blood Plasma Lipid Profiling by Ultra Performance Liquid Chromatography–Mass Spectrometry

Abstract

Exploratory or untargeted ultra performance liquid chromatography-mass spectrometry (UPLC-MS) profiling offers an overview of the complex lipid species diversity present in blood plasma. Here, we evaluate and compare eight sample preparation protocols for optimized blood plasma lipid extraction and measurement by UPLC-MS lipid profiling, including four protein precipitation methods (i.e., methanol, acetonitrile, isopropanol, and isopropanol-acetonitrile) and four liquid-liquid extractions (i.e., methanol combined with chloroform, dichloromethane, and methyl-tert butyl ether and isopropanol with hexane). The eight methods were then benchmarked using a set of qualitative and quantitative criteria selected to warrant compliance with high-throughput analytical workflows: protein removal efficiency, selectivity, repeatability, and recovery efficiency of the sample preparation. We found that protein removal was more efficient by precipitation (99%) than extraction (95%). Additionally, isopropanol appeared to be the most straightforward and robust solvent (61.1% of features with coefficient of variation (CV) < 20%) while enabling a broad coverage and recovery of plasma lipid species. These results demonstrate that isopropanol precipitation is an excellent sample preparation procedure for high-throughput untargeted lipid profiling using UPLC-MS. Isopropanol precipitation is not limited to untargeted profiling and could also be of interest for targeted UPLC-MS/MS lipid analysis. Collectively, these data show that lipid profiling greatly benefits from an isopropanol precipitation in terms of simplicity, protein removal efficiency, repeatability, lipid recovery, and coverage.

Bile Acid Profiling and Quantification in Biofluids Using Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry

“…41 High recoveries of BAs associated with high reproducibility were found in urine respectively around 92.7% and 8.58%. Recovery of the standards in plasma and serum were around 4% less than for urine recoveries, whereas reproducibility around 2% higher than for urine.…”

“…Supernatant (100 µL) was transferred to 0.5 mL Eppendorf 96-deepwell plates and 300 µL of ice-cold methanol were added to each sample. 41 All plates were heat sealed (Thermo Fisher 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Mass spectrometry parameters were as follows: capillary voltage was set at 1.5 kV, cone voltage at 60 V, source temperature 150 °C, desolvation temperature at 600 °C, desolvation gas flow at 1000 L/h, cone gas flow at 150 L/h. Bile acid species yielding characteristic fragments when subjected to collision-induced dissociation were assayed using multiple reaction monitoring (MRM), while those that did not fragment were assayed by selected ion monitoring (SIM).…”

LC–MS based global metabolite profiling: the necessity of high data quality

“…To solve these issues most researchers utilize sample preparation combined with chromatographic separation to decrease the complexity of the sample and remove substances that have a negative impact on the MS detection. A plentitude of publications have suggested various protocols for unbiased and simple sample preparation for global metabolite profiling of blood-, urine-or cell-based samples (Bruce et al 2009;Dunn et al 2011;León et al 2013;Michopoulos et al 2009;Pereira et al 2010;Rico et al 2014;Sarafian et al 2014;Tulipani et al 2013;Want et al 2010;Vorkas et al 2015;Vuckovic 2012). However, any selection of a particular approach will come with its own benefits and drawbacks in relation to the composition of the sample.…”

“…A particular focus during the last ten years has been on improving metabolite coverage (Vuckovic 2012), though little emphasis has been placed on the suitability of these protocols in regards to data quality (Sarafian et al 2014;Tulipani et al 2013Tulipani et al , 2015. The relationship between metabolite coverage and consistent reliable data is a complicated matter since these factors depend on each other.…”

Optimizing the lipidomics workflow for clinical studies—practical considerations

“…For large-scale studies, the sample preparation method should be as simple and robust as possible. A recent study demonstrated that good recoveries can also be obtained with simple protein precipitation with 2-propanol [16]. This method was compared with three other protein precipitation methods and with four extraction methods, including the modified Folch extraction and extraction with MBTE.…”