A HIGH-THROUGHPUT HPLC ASSAY FOR LEVOSIMENDAN IN
HUMAN PLASMA WITH ESI-MS/MS DETECTION
LAURIAN VLASE1, BÉLA KISS2*, CORINA BOCSAN3, SANDA NEGRUTIU4, IOAN HOREA
VLAD4, ANCA BUZOIANU3
1Department of Pharmaceutical Technology and Biopharmacy, Faculty of Pharmacy, “Iuliu Haţieganu” University of
Medicine and Pharmacy, no. 8 Victor Babeş, RO-400012, Cluj-Napoca, Romania
2Department of Toxicology, Faculty of Pharmacy, “Iuliu Haţieganu” University of Medicine and Pharmacy, no. 6 Pasteur,
RO-400023, Cluj-Napoca, Romania
3Department of Pharmacology, Faculty of Medicine, “Iuliu Haţieganu” University of Medicine and Pharmacy, no. 8 Victor
Babeş, RO-400012, Cluj-Napoca, Romania
4Cardiovascular Surgical Clinic, “Nicolae Stancioiu” Heart Institute, Cluj-Napoca Romania
*corresponding author: kbela@umfcluj.ro
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Abstract:
A new rapid, sensitive and selective liquid chromatography method coupled with ESI-MS/MS detection was developed and
validated for the quantification of levosimendan in human plasma. As a part of the optimization process, two ion sources
(electrospray ionization – ESI, and atmospheric pressure chemical ionization – APCI) were tested, in both, negative and
positive ion mode and with different mobile phases for the chromatographic separation. The final assay consisted in a
separation performed on a Zorbax SB-C18 column (100 mm x 3.0 mm i.d., 3 μm) with isocratic elution using a 66/34 (% v/v)
mixture of 0.1% acetic acid /acetonitrile as mobile phase, followed by a gradient washing step. The MS/MS detection was
performed by using ESI in negative ion mode and with m/z = 279.1→227.1 as the final transition monitored. The assay
presents several advantages, such as the small sample volume (200 μL), simple and rapid sample processing technique
(protein precipitation), high sensitivity and a very good selectivity guaranteed by the MS/MS detection. The method was
successfully applied to a pharmacokinetic study in human subjects after an infusion of L-SIM.

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