Quantitative metabolomics for dynamic metabolic engineering using stable isotope labeled internal standards mixture (SILIS)

Abstract Background: The differential diagnosis of d-2-hydroxyglutaric aciduria (d-2-HGA), l-2-hydroxyglutaric aciduria (l-2-HGA), and the combined d/l-2-hydroxyglutaric aciduria (d/l-2-HGA) can be accomplished only by the measurement of the corresponding 2-hydroxyglutarate (2-HG). Available methods for the determination of d- and l-2-HG in urine are either time-consuming and expensive or have not been extensively validated. We aimed to develop a method for their rapid and sensitive measurement. Methods: We used liquid chromatography–tandem mass spectrometry (LC-MS/MS) for the determination of d- and l-2-HG with stable-isotope-labeled internal standards. Urine samples of 20 μL were mixed with 250 μL of methanol containing the internal standards and subsequently dried under nitrogen. The analytes were derivatized by use of diacetyl-l-tartaric anhydride (DATAN) to obtain diastereomers, which were separated on an achiral C18 HPLC column and detected by MS/MS in multiple-reaction-monitoring mode. Results: The use of DATAN as chiral derivatization reagent provided very well separated peaks of the formed diastereomers of d- and l-2-HG, with a total runtime of 5 min. The inter- and intraassay CVs for d- and l-2-HG ranged from 3.4% to 6.2%. Mean recoveries of d- and l-2-HG, evaluated on two concentrations, were 94%. Detection limit of the presented method was 20 pmol for a sample volume of 20 μL. Method comparison of the LC-MS/MS method with a gas chromatography–mass spectrometry method, in which d- and l-2-HG were derivatized with R-(−)-butanol, showed good agreement between the two methods. Conclusions: Urinary d- and l-2-HG can be analyzed by MS/MS after derivatization with DATAN. The presented method may be suitable for the differential diagnosis of 2-HGA.

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The analysis of acetaminophen (paracetamol) and seven metabolites in rat, pig and human plasma by U(H)PLC–MS

Bioanalysis ◽

10.4155/bio-2020-0015 ◽

2020 ◽

Vol 12 (7) ◽

pp. 485-500

Author(s):

Rebecca Dargue ◽

Isobelle Grant ◽

Leanne C Nye ◽

Andy Nicholls ◽

Theo Dare ◽

...

Keyword(s):

Stable Isotope ◽

Sample Preparation ◽

Oral Administration ◽

Human Plasma ◽

Wistar Rats ◽

Rat Plasma ◽

Mouse Serum ◽

Internal Standards ◽

Minimal Sample

A U(H)PLC–MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 μl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 μg/ml (APAP-C), 0.58 μg/ml (APAP-SG), 0.84 μg/ml (APAP-NAC), 2.75 μg/ml (APAP-S), 3.00 μg/ml (APAP-G) and 16 μg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.

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Synthesis of deuterium labelled analogues of very long chain fatty acids C22:0, C24:0 and C26:0 for use as internal standards in stable isotope dilution analysis

International Journal of Radiation Applications and Instrumentation Part A Applied Radiation and Isotopes ◽

10.1016/0883-2889(88)90395-4 ◽

1988 ◽

Vol 39 (6) ◽

pp. 614

Author(s):

H.J. ten Brink ◽

C. Jakobs ◽

F. Bickelhaupt

Keyword(s):

Fatty Acids ◽

Stable Isotope ◽

Isotope Dilution ◽

Isotope Dilution Analysis ◽

Long Chain Fatty Acids ◽

Stable Isotope Dilution Analysis ◽

Internal Standards ◽

Stable Isotope Dilution ◽

Long Chain ◽

Chain Fatty Acids

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Comparison of Heavy Labeled (SIL) Peptide versus SILAC Protein Internal Standards for LC-MS/MS Quantification of Hepatic Drug Transporters

International Journal of Proteomics ◽

10.1155/2014/451510 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 16

Author(s):

Bhagwat Prasad ◽

Jashvant D. Unadkat

Keyword(s):

Stable Isotope ◽

Standard Method ◽

Drug Transporters ◽

Organic Anion ◽

Internal Standard ◽

Trypsin Digestion ◽

Internal Standard Method ◽

Internal Standards ◽

Lysine Residues ◽

Human Livers

We studied the precision of quantification of organic anion-transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, and P-glycoprotein (P-gp) in human livers by surrogate peptide based LC-MS/MS approach using two different internal standards: stable isotope labeled peptide (SIL) versus stable isotope labeled protein (SILAC). The SIL peptides were procured commercially and the SILAC proteins were generated in-house by labeling arginine and/or lysine residues in cells expressing these transporters. Liver tissue n=20 was homogenized and the membrane fraction was isolated. The membranes were trypsin digested and the peptides were analyzed using LC-MS/MS under optimized conditions. The precision in the quantification of proteins in three independently trypsin digested samples from each liver was calculated as the standard deviation of the log transformed protein concentration. The precision of the SIL internal standard method was either slightly (P<0.05, paired t-test) better than that of the SILAC method (OATP1B1, OATP1B3, and P-gp) or not different (OATP2B1). Trypsin digestion, as measured by the response of the labeled peptide derived from the SILAC protein, was consistent across liver samples. These results indicate that when maximum trypsin digestion is ensured, the SIL internal standard method can be used with confidence for quantification of drug transporters.

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