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Development and validation of a liquid chromatography/tandem mass spectrometry method for the simultaneous quantification of serotonin and thromboxane B2 from activated platelets
Author(s) -
Minet Valentine,
Evrard Jonathan,
Vancraeynest Christelle,
Dogné JeanMichel,
Mullier François,
Pochet Lionel
Publication year - 2018
Publication title -
international journal of laboratory hematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.705
H-Index - 55
eISSN - 1751-553X
pISSN - 1751-5521
DOI - 10.1111/ijlh.12901
Subject(s) - chromatography , chemistry , electrospray ionization , platelet , tandem mass spectrometry , mass spectrometry , selected reaction monitoring , detection limit , medicine
Availability of a rapid and reliable platelet activation assay avoiding limitations of current techniques would be valuable to diagnose heparin‐induced thrombocytopenia and platelet secretion disorders. Objectives The first aim was to develop and validate an ultra‐performance liquid chromatography‐tandem mass spectrometry ( UHPLC ‐ MS / MS ) method to quantify in a single run TxB2 synthesized and serotonin released from platelets. The second aim was to use our method in association with light transmission aggregometry ( LTA ) to select good platelet responders for the diagnosis of HIT . Methods Electrospray ionization and chromatographic separation were optimized for the simultaneous dosage of serotonin and TxB2. The method was validated according to the European Medicines Agency ( EMA ) guideline for bioanalytical method validation. LTA was performed with monoclonal anti‐CD9 (clone ALB 6) as platelet activator to select good responders. Results Detection was performed using a tandem mass spectrometer with alternated positive and negative electrospray ionization. The total run time was 6 minutes. The method was validated for calibration curves, precision, accuracy, lower limit of quantification, carry‐over, selectivity, and matrix effect. Platelet response to ALB6 was highly variable among donors. Conclusion We developed and validated a UHPLC ‐ MS / MS method for the simultaneous quantification of TxB2 and serotonin.