High-Throughput Analysis of Underivatized Amino Acids and Acylcarnitines in Infant Serum: A Micromethod Based on Stable Isotope Dilution Targeted HILIC-ESI-MS/MS

Abstract

Amino acids and acylcarnitines are important biomarkers of the body’s energy state and can be used as diagnostic markers of certain inborn errors of metabolism. Few multianalyte methods for high-throughput analysis in serum exist for these compounds, but micromethods suitable for use in young children and infants are lacking. Therefore, we developed a quantitative high-throughput multianalyte hydrophilic interaction liquid chromatography–tandem mass spectrometry method preceded by a derivatization-free sample preparation using minimum amounts of serum (25 μL). Isotopically labeled standards were utilized for quantification. Forty amino acids and amino acid derivatives and 22 acylcarnitines were detected by applying a multiple reaction monitoring mode within a 20 min run. The method was comprehensively validated, comprising linearity, accuracy, (intraday/interday) precision, and quantitation limits, of which the latter ranged from 0.25 to 50 nM for acylcarnitines and from 0.005 to 1 μM for amino acids and their derivatives. Application of the method to 145 serum samples of three- to four-month-old healthy infants showed excellent reproducibility for multiday analyses and enabled simultaneous amino acid and acylcarnitine profiling in this age group.

A 14-Day Double-Blind, Randomized, Controlled Cross-Over Intervention Study With Anti-bacterial Benzyl Isothiocyanate From Nasturtium (Tropaeolum majus) on Human Gut Microbiome and Host Defense

“…Using PCA, we found that samples were clustered by individuals rather than by time of sampling, which could suggest stability of the individual microbiome throughout intervention and high inter-individual variation in microbiome composition (Figure 2A). Walker et al [32] also observed this in a clinical trial of 14 overweight men on different controlled diets containing indigestible starch or non-starch polysaccharides. They suggested that this could be associated with inter-individual different responses of the gut microbiota to dietary changes [32].…”

“…Walker et al [32] also observed this in a clinical trial of 14 overweight men on different controlled diets containing indigestible starch or non-starch polysaccharides. They suggested that this could be associated with inter-individual different responses of the gut microbiota to dietary changes [32]. There were also no significant differences between baseline samples, which could be indicative of the absence of a carryover effect between the two intervention phases.…”

“…Targeted analysis of amino acids and amino acid derivates was carried out as previously reported [32] utilizing a QTRAP 6500+ mass spectrometer (Sciex, Darmstadt, Germany) operated in positive ionization mode (ESI+), with an ion spray voltage of +5500 V. Nitrogen served as the curtain gas at 35 psi, while gas 1 and gas 2 were set at 55 psi and 65 psi, respectively. The source temperature was maintained at 450 °C.…”

A 14-Day Double-Blind, Randomized, Controlled Crossover Intervention Study with Anti-Bacterial Benzyl Isothiocyanate from Nasturtium (Tropaeolum majus) on Human Gut Microbiome and Host Defense

“…Targeted analysis of amino acids and amino acid derivates was carried out as previously reported [32] utilizing a QTRAP 6500+ mass spectrometer (Sciex, Darmstadt, Germany) operated in positive ionization mode (ESI+), with an ion spray voltage of +5500 V. Nitrogen served as the curtain gas at 35 psi, while gas 1 and gas 2 were set at 55 psi and 65 psi, respectively. The source temperature was maintained at 450 • C. Multiple reaction monitoring (MRM) mode was utilized, with scheduled detection windows tailored to each metabolite.…”

Target and Semitarget Analysis of Advanced Glycation End Products Using a New Pair of Permanently Positively Charged Stable Isotope Labeling Agents

“…Some AGEs exhibit fluorescence property, owing to which fluorescence method has been used to monitor them; however, distinguishing analytes from interferences is difficult . High-performance liquid chromatography (HPLC) and HPLC-tandem mass spectrometry (HPLC-MS/MS) are the most often used methods for AGE detection. ,, AGEs can be separated from interferences, and the selectivity can be considerably improved. However, the matrix effect has been frequently reported to influence the accuracy.…”