First Dimension Trap-and-Elute Combined with Second Dimension
Reversed-Phase Liquid Chromatography Separation Using a
Two-Dimensional-Liquid Chromatography-Tandem Mass Spectrometry
System for Sensitive Quantification of Human Insulin and Six
Insulin Analogs in Plasma: Improved Chromatographic Resolution
and Stability Testing

J 2025

First Dimension Trap-and-Elute Combined with Second Dimension Reversed-Phase Liquid Chromatography Separation Using a Two-Dimensional-Liquid Chromatography-Tandem Mass Spectrometry System for Sensitive Quantification of Human Insulin and Six Insulin Analogs in Plasma: Improved Chromatographic Resolution and Stability Testing

SISTIK, Pavel; Romana URINOVSKA; Klara HANDLOSOVA; Petr HANDLOS; Katerina ANDELOVA et. al.

Basic information

Original name

First Dimension Trap-and-Elute Combined with Second Dimension Reversed-Phase Liquid Chromatography Separation Using a Two-Dimensional-Liquid Chromatography-Tandem Mass Spectrometry System for Sensitive Quantification of Human Insulin and Six Insulin Analogs in Plasma: Improved Chromatographic Resolution and Stability Testing

Authors

SISTIK, Pavel (203 Czech Republic); Romana URINOVSKA (203 Czech Republic); Klara HANDLOSOVA (203 Czech Republic); Petr HANDLOS (203 Czech Republic); Katerina ANDELOVA (203 Czech Republic); Jan JUŘICA (203 Czech Republic, belonging to the institution) and David STEJSKAL (203 Czech Republic)

Edition

Journal of Separation Science, Weinheim, Wiley, 2025, 1615-9306

Other information

Language

English

Type of outcome

Article in a journal

Field of Study

30104 Pharmacology and pharmacy

Country of publisher

Germany

Confidentiality degree

is not subject to a state or trade secret

References:

URL

Impact factor

Impact factor: 2.800 in 2023

Organization unit

Faculty of Medicine

DOI

http://dx.doi.org/10.1002/jssc.70092

UT WoS

001416960300001

EID Scopus

2-s2.0-85218343396

Keywords in English

two-dimensional liquid chromatography; tandem mass spectrometry; insulin quantification; plasma analysis; chromatographic resolution

Abstract

V originále

Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 µm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 µm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50–15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.

Cite

SISTIK, Pavel; Romana URINOVSKA; Klara HANDLOSOVA; Petr HANDLOS; Katerina ANDELOVA; Jan JUŘICA and David STEJSKAL. First Dimension Trap-and-Elute Combined with Second Dimension Reversed-Phase Liquid Chromatography Separation Using a Two-Dimensional-Liquid Chromatography-Tandem Mass Spectrometry System for Sensitive Quantification of Human Insulin and Six Insulin Analogs in Plasma: Improved Chromatographic Resolution and Stability Testing. Journal of Separation Science. Weinheim: Wiley, 2025, vol. 48, No 2, p. 1-16. ISSN 1615-9306. Available from: https://dx.doi.org/10.1002/jssc.70092.

@article{2476874,
   author = {Sistik, Pavel and Urinovska, Romana and Handlosova, Klara and Handlos, Petr and Andelova, Katerina and Juřica, Jan and Stejskal, David},
   article_location = {Weinheim},
   article_number = {2},
   doi = {http://dx.doi.org/10.1002/jssc.70092},
   keywords = {two-dimensional liquid chromatography; tandem mass spectrometry; insulin quantification; plasma analysis; chromatographic resolution},
   language = {eng},
   issn = {1615-9306},
   journal = {Journal of Separation Science},
   title = {First Dimension Trap-and-Elute Combined with Second Dimension Reversed-Phase Liquid Chromatography Separation Using a Two-Dimensional-Liquid Chromatography-Tandem Mass Spectrometry System for Sensitive Quantification of Human Insulin and Six Insulin Analogs in Plasma: Improved Chromatographic Resolution and Stability Testing},
   url = {https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jssc.70092},
   volume = {48},
   year = {2025}
}

TY  - JOUR
ID  - 2476874
AU  - Sistik, Pavel - Urinovska, Romana - Handlosova, Klara - Handlos, Petr - Andelova, Katerina - Juřica, Jan - Stejskal, David
PY  - 2025
TI  - First Dimension Trap-and-Elute Combined with Second Dimension Reversed-Phase Liquid Chromatography Separation Using a Two-Dimensional-Liquid Chromatography-Tandem Mass Spectrometry System for Sensitive Quantification of Human Insulin and Six Insulin Analogs in Plasma: Improved Chromatographic Resolution and Stability Testing
JF  - Journal of Separation Science
VL  - 48
IS  - 2
SP  - 1-16
EP  - 1-16
PB  - Wiley
SN  - 16159306
KW  - two-dimensional liquid chromatography
KW  - tandem mass spectrometry
KW  - insulin quantification
KW  - plasma analysis
KW  - chromatographic resolution
UR  - https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jssc.70092
N2  - Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 µm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 µm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50–15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.
ER  -

SISTIK, Pavel; Romana URINOVSKA; Klara HANDLOSOVA; Petr HANDLOS; Katerina ANDELOVA; Jan JUŘICA and David STEJSKAL. First Dimension Trap-and-Elute Combined with Second Dimension Reversed-Phase Liquid Chromatography Separation Using a Two-Dimensional-Liquid Chromatography-Tandem Mass Spectrometry System for Sensitive Quantification of Human Insulin and Six Insulin Analogs in Plasma: Improved Chromatographic Resolution and Stability Testing. \textit{Journal of Separation Science}. Weinheim: Wiley, 2025, vol.~48, No~2, p.~1-16. ISSN~1615-9306. Available from: https://dx.doi.org/10.1002/jssc.70092.

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