Peripheral venous blood and urine samples in case 1 were collected by medical professionals at the hospital site. In the post-mortem cases 2, 3, and 4, femoral venous blood and urine samples were collected during the medico-legal autopsy. All samples were stored at –20 °C until analysis. Urine screenings were performed by CEDIA for common drugs on an Indiko Plus device and by an untargeted LC-MS screening method as described elsewhere [26 (link)]. The system consisted of a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fisher, San Jose, CA, USA) coupled to an ABSciex 3200 QTrap linear ion trap quadrupole mass spectrometer (ABSciex, Darmstadt/Germany). Automated MS matching was performed against a commercially available database [27 ]. Positive results were confirmed in blood by a subsequent confirmatory LC-MS/MS according to a previously validated method [28 (link)]. The system consisted of a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fisher, San Jose, CA, USA) coupled to an ABSciex 3200 QTrap linear ion trap quadrupole mass spectrometer (ABSciex, Darmstadt/Germany).
Thermo fischer ultimate 3000 uhplc system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Fisher Scientific Ultimate 3000 UHPLC system is a high-performance liquid chromatography instrument designed for advanced separation and analysis applications. It features a compact design, precise flow control, and high-resolution detection capabilities to deliver reliable and consistent results.
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5 protocols using thermo fischer ultimate 3000 uhplc system
1
Comprehensive Toxicology Screening Protocol
2
HRMS Analysis of GHB Conjugates
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HRMS measurements of synthesized GHB conjugates (1 μg/ml in water [amino acid conjugates]) or MeOH (fatty acid conjugates) were performed on a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fischer Scientific, San Jose, CA, USA) coupled to a HR quadrupole time of flight (qTOF) instrument system (TripleTOF 6600, Sciex, Concord, ON, Canada) as described in detail elsewhere.10 , 21 , 22 Briefly, two different columns—reversed phase (RP) (Waters XSelect HSST RP‐C18 column, Waters, Baden‐Daettwil, Switzerland [150 mm × 2.1 mm, 2.5 μm particle size]) and hydrophilic interaction liquid chromatography (HILIC) (Merck SeQuant ZIC HILIC column, Merck, Darmstadt, Germany [150 mm × 2.1 mm, 3.5 μm particle size])—were used for chromatographic separation. HRMS (mass range m/z 100–1000) and MS/MS (mass range m/z 50–1000) data were acquired by information‐dependent data acquisition (IDA; top 4) in positive and negative ionization mode (resolving power 30,000 in MS and 15,000 in MS/MS).
3
Targeted Metabolomics Analysis Pipeline
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MS measurements were performed in randomized order in three batches (serum, urine Ut1, urine Ut2) on a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fischer Scientific, San Jose, CA, USA) coupled to a HR TOF instrument system (TripleTOF 6600, Sciex, Concord, ON, Canada) as described elsewhere and in the supplementary information [13 (link),23 (link),24 (link)]. Briefly, two different columns—RP (Waters XSelect HSST RP-C18 column, Waters, Baden-Daettwil, Switzerland) (150 mm × 2.1 mm, 2.5 µm particle size)) and HILIC (Merck SeQuant ZIC HILIC column, Merck, Darmstadt, Germany) (150 mm × 2.1 mm, 3.5 µm particle size)) were used for chromatographic separation. HR MS and MS/MS data were acquired by two methods: TOF MS only and information dependent data acquisition (IDA) separated in positive and negative ionization mode (resolving power 30,000 in MS and 15,000 in MS/MS). A SST described in detail in reference [24 (link)] was measured after every fifth sample and was checked for reproducibility of the data by retention time (RT) shifts and peak area comparison using MultiQuant V 2.1 (Sciex, Concord, ON, Canada). Further, a pooled QC sample was additionally measured after every fifth sample.
4
Cannabinoid Quantification in Blood
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Given the undetectability of cannabinoids in the screening analysis, all blood samples were subjected to targeted cannabis analysis on tetrahydrocannabinol (THC), cannabidiol (CBD) and the THC metabolites hydroxy-THC (THC-OH) and THC carboxylic acid (THC-COOH). LOQs were as follows: 0.5 ng/mL, 0.2 ng/mL, 0.5 ng/mL, and 5 ng/mL for THC, CBD, THC-OH, and THC-CCOH, respectively. Sample extracts were analyzed on a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fischer Scientific) coupled to a Sciex 5500 QTrap linear ion trap quadrupole mass spectrometer (Sciex). Details on the method can be found in the Supplementary Material. MS mode, QC, and data evaluation were performed as described above for the drug quantification.
5
Targeted Quantitative Analysis of Drugs
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Sample preparation and targeted quantitative analysis for drugs (of abuse) were carried out with a previously validated method according to Staeheli et al. (83 analytes in 11 postmortem matrices including diazepam, nordiazepam, morphine, codeine, mirtazapine and citalopram; details regarding lower limits of quantification and corresponding calibration ranges see Table S3 in the supplementary material ; calibration in whole blood) [19 (link)]. In brief, a two-step liquid-liquid extraction (LLE) was used to extract 20 µL femoral blood (butyl acetate/ethyl acetate (1:1, v/v) at pH 7.4 and pH 13.5). The extracts were combined, evaporated to dryness (N2) and reconstituted in mobile phase (60 µL; eluent A:B (90:10, v/v); eluent A: 10 mM ammonium formate buffer in water with 0.1% (v/v) formic acid; eluent B: acetonitrile with 0.1% (v/v) formic acid). The analysis was conducted on a Thermo Fischer Ultimate 3000 UHPLC system (Thermo Fischer, San Jose, CA, USA) coupled to a Sciex 5500 QTrap linear ion trap quadrupole mass spectrometer (Sciex, Darmstadt, Germany) with instrument settings according to Staeheli et al. [19 (link)]. Samples from the same case were prepared and analyzed within the same batch.
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