scholarly journals The use of LC tandem mass spectrometry as part of a workflow for the screening and identification of hemoglobin variants. Characterization of Hb Ullevaal as an example

2017 ◽  
Vol 41 (3) ◽  
Author(s):  
Matthias Weber ◽  
Julia J.M. Eekels
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Cation Exchange ◽  
Peripheral Blood ◽  
Globin Gene ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
South Florida ◽  
Tandem Mass ◽  
Genetic Sequencing

AbstractBackground:About 2/3 of the hemoglobin (Hb) variants do not show a charge difference to the wildtype entity but most of them differ in hydrophobicity. In addition to cation exchange chromatography, globin differentiation by liquid chromatography-tandem mass spectrometry (MS) was introduced. Hb Ullevaal was chosen as one example to demonstrate the performance of the approach.Methods:Screening for Hb variants was performed using cation exchange HPLC. For globin separation reversed phase-LC/MS was performed. Tryptic digests of variants were separated on RP-HPLC with or without CID-fragmentation and database search for identification of mutation bearing fragments. Sequencing of the β-globin gene has been performed.Results:HbS, HbC, HbE, Hb South Florida and Hb Ullevaal show typical and distinct patterns in the globin LC/MS according to the theoretical protein data. The tryptic digest of Hb Ullevaal resulted in the identification of the respective mutated peptide βT9, which was confirmed by genetic sequencing.Conclusions:By the application of globin-LC/MS two more dimensions for the Hb identification are added, hydropathicity and protein mass. With this workflow as screening procedure for Hb variants it is expected to be able to detect and identify the majority of variants with the exception of highly unstable variants, which cannot be determined in the peripheral blood at all. A negative result makes the presence of a significant Hb variant in the peripheral blood improbable.

scholarly journals Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2123
Author(s):  
Luboš Fical ◽  
Maria Khalikova ◽  
Hana Kočová Vlčková ◽  
Ivona Lhotská ◽  
Zuzana Hadysová ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Solid Phase Extraction ◽  
Solid Phase ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Tandem Mass ◽  
Phase Extraction ◽  
Hydrophilic Interaction

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.

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