Journal of Pharmaceutical and Biomedical Analysis 21 (1999) 1045–1051 Liquid chromatographic determination of urinary 5-methyl-2-deoxycytidine and pseudouridine as potential biological markers for leukaemia Carlo Giorgio Zambonin a , Antonella Aresta b , Francesco Palmisano b, *, Giorgina Specchia c , Vincenzo Liso c a Dipartimento di Chimica, Uniersita ` degli Studi della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy b Dipartimento di Chimica, Uniersita ` degli Studi di Bari, Via Orabona 4, 70126 Bari, Italy c Cattedra di Ematologia, Uniersita ` degli Studi di Bari, Piazza G. Cesare 11, 70100 Bari, Italy Received 16 April 1999; received in revised form 30 July 1999; accepted 21 August 1999 Abstract A simple reversed-phase liquid chromatographic (LC) method for the determination of urinary 5-methyl-2-deoxy- cytidine (m 5 dCyd), recently claimed (on the basis of an imuno-technique) to be a potential marker for leukaemia, has been developed. Sample pre-treatment is based on a microcolumn clean-up step with an average recovery of 79% and a RSD of 3%. Detection limit was 0.2 g/ml which is about tenfold lower than levels previously measured by an ELISA method in urine of healthy individuals. The creatinine (Cre) excretion, necessary for normalising the m 5 dCyd excretion, was evaluated by ion-pair liquid chromatography which permitted the simultaneous determination of pseudouridine (), a modiﬁed nucleoside also potentially useful as a marker for leukaemia. The described LC procedures were applied to the analysis of urine samples from healthy individuals and leukaemia patients. While the urinary /Cre ratio was found signiﬁcantly increased for leukaemia patients, the urinary m 5 dCyd levels in healthy individuals were below the detection limits and did not increase in presence of the malignant disease. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Liquid chromatography; Biological markers; Leukaemia www.elsevier.com/locate/jpba 1. Introduction A marker for a malignant disease is deﬁned as any substance that is elevated in the serum or urine, reﬂecting the presence of the malignant process. Ideally this substance should be present early enough and in sufﬁcient quantity to allow screening for the disease; the quantity produced should also reﬂect the bulk of malignancy and ﬁnally the level of marker should reﬂect the suc- cess of therapy [1]. * Corresponding author. Tel.: +39-080-544-2016; fax. + 39-080-544-2026. E-mail address: palmisano@chimica.uniba.it (F. Palmisano) 0731-7085/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII:S0731-7085(99)00221-6