The application of internal standards in quantitative and qualitative bioanalysis is a commonly used procedure. They are usually isotopically labeled analogs of the analyte, used in quantitative LC-MS analysis. Usually, ²H, ¹³C, ¹⁵N and ¹⁸O isotopes are used. The synthesis of deuterated isotopologues is relatively inexpensive, however, due to the isotopic effect of deuterium and the lack of isotopologue co-elution, usually they are not considered as good internal standards for LC-MS quantification. On the other hand, the preparation of ¹³C, ¹⁵N and ¹⁸O containing standards of drugs and their metabolites requires a complicated multistep de novo synthesis, starting from the isotopically labeled substrates, which are usually expensive. Therefore, there is a strong need for the development of low-cost methods for isotope-labeled standard preparations for quantitative analysis by LC-MS. The presented review concentrates on the preparation of deuterium-labeled standards by hydrogen−deuterium exchange reactions at the carbon centers. Recent advances in the development of the methods of isotopologues preparation and their application in quantitative analysis by LC-MS are evaluated.

The metabolomics and proteomics analysis of saliva, an excellent biofluid that is a rich source of biological compounds, allows for the safe and frequent screening of drugs, their metabolites, and molecular biomarkers of various diseases. One of the most frequently used analytical methods in saliva analysis is liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry. The low ionisation efficiency of some compounds and a complex matrix makes their identification by MS difficult. Furthermore, quantitative analysis by LC-MS frequently cannot be performed without isotopically labelled standards, which usually have to be specially synthesised. This review presented reports on qualitative and quantitative approaches in salivary metabolomics and proteomics. The purpose of this manuscript was to present the challenges, advances, and future prospects of mass spectrometry, both in the analysis of salivary metabolites and proteins. The presented review should appeal to those interested in the recent advances and trends in qualitative and quantitative mass spectrometry in salivary metabolomics and proteomics, which may facilitate a diagnostic accuracy, the evaluation of treatment efficacy, the early diagnosis of disease, and a forensic investigation of some unapproved drugs for any medical or dietary administration.

Armodafinil, the R enantiomer of modafinil, was approved in 2007 by the US Food and Drug Administration as a wake-promoting agent for excessive sleepiness treatment. Due to its abuse by students and athletes, there is a need of its quantification. Quantitative analysis by liquid chromatography-mass spectrometry, however, though very common and sensitive, frequently cannot be performed without isotopically labeled standards which usually have to be specially synthesized. Here we reported our investigation on the preparation of deuterated standard of armodafinil based on the simple and inexpensive hydrogen–deuterium exchange reaction at the carbon centers. The obtained results clearly indicate the possibility of introduction of three deuterons into the armodafinil molecule. The introduced deuterons do not undergo back exchange under neutral and acidic conditions. Moreover, the deuterated and non-deuterated armodafinil isotopologues revealed co-elution during the chromatographic analysis. The ability to control the degree of deuteration using different reaction conditions was determined. The proposed method of deuterated armodafinil standard preparation is rapid, cost-efficient and may be successfully used in its quantitative analysis by LC-MS.

Early detection of any preeclampsia biomarkers may lower the risk of mortality, both for a mother and a child. Our study focuses on techniques for preeclampsia biomarker identification by comparing the results of a method using liquid chromatography mass spectrometry in multiple reaction monitoring mode (LC-MS/MS) with those by the enzyme-linked immunosorbent assay (ELISA) test, as well as by comparing the obtained results with clinical data. In the proposed LC-MS/MS method a tryptic digest peptide charge derivatization strategy was used as a tool for sensitive detection of podocin, i.e., a previously discovered preeclampsia biomarker present in urine samples from pregnant women. Urine samples from pregnant women with diagnosed preeclampsia were collected at different stages of pregnancy and from healthy subjects, and then were analyzed by ELISA test and the proposed method with LC-MS/MS. Charge derivatization of the ε amino group of C-terminal lysine residues in tryptic digests by 2,4,6-triphenylpyrylium salt was performed to increase the ionization efficiency in the LC-MS/MS mode. Podocin was identified at the early stage of pregnancy, while its detection using an ELISA test was not possible. The protocol for urine sample preparation was optimized. Our results show that the proposed method by LC-MS/MS in combination with peptide charge derivatization, provides an ultrasensitive tool for diagnosis of preeclampsia, and provides earlier detection than a clinical diagnosis or ELISA test. The proposed solution may revolutionize medical diagnostics.

This review presents numerous studies in which mass spectrometry has been used to assist forensic investigation. Due to its unique capabilities, mainly high-resolution mass data and structural information, high sensitivity, and cooperation with separation techniques, this method provides access to many tools streamlining and accelerating sample analysis. Low analyte consumption, advanced derivatization procedures and availability of isotopically labeled standards offer opportunities to study materials previously not considered viable evidence, opening new avenues in forensic investigations.

Specific and nonspecific non-covalent molecular association of biomolecules is characteristic for electrospray-ionization mass spectrometry analysis of biomolecules. Understanding the interaction between two associated molecules is of significance not only from the biological point of view but also gas phase analysis by mass spectrometry. Here we reported a formation of non-covalent dimer of quaternary ammonium denatonium cation with +1 charge detected in the positive ion mode electrospray ionization mass spectrometry analysis of denatonium benzoate. Hydrogen deuterium exchange of amide and carbon-bonded hydrogens revealed that charge neutralization of one denatonium cation is the consequence of amide hydrogen dissociation. DFT (Density Functional Theory) calculations proved high thermodynamic stable of formed dimer stabilized by the short and strong N..H-N hydrogen bond. The signal intensity of the peak characterizing non-covalent dimer is low intensity and does not depend on the sample concentration. Additionally, dimer observation was found to be instrument-dependent. The current investigation is the first experimental and theoretical study on the quaternary ammonium ions dimer. Thus the present study has great significance for understanding the structures of the biomolecules as well as materials.