Analytical considerations for (un)‐targeted metabolomic studies with special focus on forensic applications

Abstract

Over the past few years, the interest in metabolomics has increased in various fields including forensic toxicology. Forensic analysis typically requires a high degree of accuracy, which is often a problem in metabolomics applications. We aimed for a systematic evaluation of different analytical considerations of a metabolomics workflow allowing a targeted approach within an untargeted setup. Samples with 69 metabolites from different chemical classes were qualitatively and quantitatively analyzed on a high resolution quadrupole time of flight mass spectrometer coupled to liquid chromatography (UHPLC-QTOF). Three issues were addressed: (a) Two different approaches on "blind matrix" a simulated body fluid (SBF) and plasma-filtrate, were tested for calibration samples; (b) comparison of two different HPLC columns, reverse-phase (RP) and hydrophilic interaction chromatography (HILIC); and (c) comparison of three different acquisition modes (TOF-MS, information dependent data acquisition (IDA), and sequential window acquisition of all theoretical fragment-ion spectra (SWATH). Samples were measured repeatedly for method comparison based on sensitivity, accuracy, precision, and detection robustness. The blind matricesshowed similar accuracy for most analytes, while SBF provided an easier preparation with satisfying results. To cover a wide part of the human metabolome, a combination of RP and HILIC showed the best results. The different scan modes performed equally regarding metabolite quantification while TOF-MS was more sensitive but lacked MS/MS spectra generation. IDA and SWATH files were aligned to various databases where IDA showed good MS/MS spectra matches. SWATH seemed to be beneficial in detection rate but was incompatible with many important software tools in metabolomics.

Identification of new urinary gamma‐hydroxybutyric acid markers applying untargeted metabolomics analysis following placebo‐controlled administration to humans

“…Urine samples of the experimental nights after placebo and GHB intake of 19 participants (sample of one participant was unavailable) were thawed at room temperature and vortexed for 20 seconds. Forty μL of the IS mix (adenosine ribose‐D 1 15 μmol/L, arginine‐ 13 C 6 300 μmol/L, caffeine 3‐methyl‐ 13 C 200 μmol/L, carnitine trimethyl‐D 9 100 μmol/L, creatinine N‐methyl‐D 3 500 μmol/L, deoxycholic acid‐D 4 1.8 μmol/L, D‐fructose 13 C 120 μmol/L, glycine‐ 13 C 2 800 μmol/L, glycocholic acid‐D 4 150 μmol/L, hippuric acid 15 N 500 μmol/L, kynurenine‐D 4 8 μmol/L, leucine‐D 10 300 μmol/L, lysine‐D 4 700 μmol/L, phenylalanine‐D 1 300 μmol/L, proline 15 N 700 μmol/L, serine‐D 3 450 μmol/L, tryptophan‐D 5 250 μmol/L and uric acid‐ 15 N 2 500 μmol/L) was placed into autosampler filter vials (0.45 μm PTFE, Thomson Instrument Company, Oceanside, CA, USA) and 200 μL of urine sample were added. Depending on the acquisition method either 200 μL of a 1:1 (v/v) mixture of eluents A and B (for measurement on the HSST column) or C and D (for measurement on the HILIC column, see Section ) was added.…”

“…24,25 Two different chromatographic systemsreversed phase (HSST) and HILICin both positive and negative ionization mode were chosen to cover as many small molecules as possible. The applied screening method has been extensively examined recently on analyte selection, detectability and sensitivity 20 and was already successfully applied to other untargeted and targeted studies of the metabolome. [26][27][28] For peak picking and deconvolution,…”

Towards Best Practice in Hair Metabolomic Studies: Systematic Investigation on the Impact of Hair Length and Color

“…The study data were obtained by untargeted data acquisition methods using LC-HRMS with two chromatographic columns in the positive and negative ionization modes as commonly applied in untargeted metabolomic studies [ 2 ]. All samples were analyzed as described in previously published studies [ 6 , 22 ] and in randomized order on a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fischer Scientific, San Jose, CA), coupled to a high-resolution (HR) time-of-flight (TOF) instrument system (TripleTOF 6600, Sciex, Concord, ON, Canada). Methodological details and performance evaluation have been described in detail elsewhere [ 22 ].…”

“…All samples were analyzed as described in previously published studies [ 6 , 22 ] and in randomized order on a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fischer Scientific, San Jose, CA), coupled to a high-resolution (HR) time-of-flight (TOF) instrument system (TripleTOF 6600, Sciex, Concord, ON, Canada). Methodological details and performance evaluation have been described in detail elsewhere [ 22 ]. In brief, chromatographic separation was achieved by using reversed-phase (Xselect HSST RP-C18 (150 mm × 2.1 mm i.d.…”

“…For column equilibration, a series of QC Pools was injected at the sequence beginning and then repeatedly after every 5th sample. A system suitability test (SST) was prepared according to previously published protocols [ 22 , 23 ] and was injected every 10th sample to check data reproducibility and correct for any retention time (RT) shifts. Targeted peak area integration of SST analytes and endogenous compounds was performed by peak integration of precursor ions from TOF-MS runs using Multiquant V 2.1 (Sciex, Concord, ON, Canada).…”