Elsevier

Journal of Chromatography B

Volumes 949–950, 15 February 2014, Pages 58-62
Journal of Chromatography B

Short Communication
HPLC-UV analysis of thymidine and deoxyuridine in plasma of patients with thymidine phosphorylase deficiency

https://doi.org/10.1016/j.jchromb.2014.01.003Get rights and content

Highlights

  • This is the first validated method for plasma analysis of dThd and dUrd in MNGIE.

  • Plasma pretreatment by deproteinization and isocratic HPLC are the main advantages.

  • The HPLC-UV allows this application in laboratories lacking sophisticated equipment.

Abstract

We present a simple, fast and validated method for the determination of the two nucleosides thymidine (dThd) and deoxyuridine (dUrd) in plasma of patients with symptoms suggestive of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), using high performance liquid chromatography coupled with ultraviolet spectrophotometric detection (HPLC-UV). Plasma sample (100 μL) pretreatment was based on simple deproteinization by 1.2 M perchloric acid, using theophylline as internal standard (I.S.). HPLC-UV analysis was carried out on a Synergi 4 μm Hydro-RP, 150 × 4 mm I.D. column, at room temperature. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (20 mM, pH 4.5) and acetonitrile (95:5, v/v), at an isocratic flow rate of 0.7 mL/min. The UV detector was set at 267 nm. The chromatographic run lasted 19 min. Similar pyrimidine nucleotides and nucleosides do not interfere with the assay. Calibration curves were linear for both dThd and dUrd over a range of 0.5 to 5.0 μg/mL. The limit of quantitation was 0.5 μg/mL for both nucleosides and the absolute recovery was >90% for dThd, dUrd and the I.S. Both intra- and inter-assay precision and accuracy were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of dThd and dUrd in two MNGIE patients. This assay simplifies both plasma pretreatment and chromatographic conditions of previously reported procedures and describes the first validated method for the determination of the two nucleotides in human plasma.

Introduction

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disorder clinically characterized by ptosis and ophthalmoparesis, gastrointestinal dysmotility, peripheral neuropathy, leukoencephalopathy, and mitochondrial dysfunctions. MNGIE is due to mutations in TYMP gene, which encodes thymidine phosphorylase (TP) [1], [2].

The prevalence of MNGIE is currently unknown: in the last 12 years, since the identification of TYMP mutations as causative of MNGIE, the largest cohort of 102 patients was recently published [3] and a few other scattered reports are available from literature suggesting that the total number of patients worldwide is possibly less than 200.

TYMP mutations cause loss of function of the enzyme, leading to an unbalanced metabolism of thymidine (dThd) and deoxyuridine (dUrd) and to their systemic accumulation in body fluids and tissues. Reported plasma concentrations of dThd and dUrd in MNGIE patients range from 0.9 to 4.3 μg/mL and 1.25 to 5.6 μg/mL, respectively [4], [5], [6], while they are undetectable (<0.01 μg/mL) in healthy controls. Analyzing plasma dThd and dUrd concentration is the easiest way to test for TP dysfunction and support TP activity assay [4], [5], [6].

In literature only a few papers described methodologically the determination of dThd and dUrd in plasma of patients with altered thymidine metabolism, based on HPLC-UV detection [4], [7], or tandem mass spectrometry [8], involving time-consuming chromatographic gradient elution procedures to eliminate interferences [4], [7], complex sample pre-treatment [7], and lacking analytical validation.

Here we propose a validated HPLC-UV assay for the determination of dThd and dUrd in plasma of MNGIE patients, which simplifies both plasma pretreatment and chromatographic conditions of previous methods.

Section snippets

Reagents and standards

Thymidine, deoxyuridine, the internal standard (I.S.) theophylline and thymine, cytosine, uric acid, cytidine, uridine, xanthine, inosine, guanosine, tryptophan, adenosine were purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC grade acetonitrile, 12 M perchloric acid and potassium dihydrogen phosphate were purchased from Merck (Darmstadt, Germany). Ultrapure water was obtained from a MilliQ Gradient A10 apparatus (Merck Millipore, Darmstadt, Germany).

Frozen, drug-free plasma (blank plasma)

Results and discussion

The mixture of mobile phase (potassium dihydrogen phosphate buffer, 20 mM, pH 4.5 and acetonitrile, 95:5, v/v) combined with a Synergi 4 μm Hydro-RP column 150 × 4.6 mm at a flow rate of 0.7 mL/min allowed a good separation of dThd and dUrd with mean ± S.D. (n = 6) of 4.0 ± 0.1 min for dUrd, 6.9 ± 0.1 min for dThd and 18.9 ± 0.6 min for the I.S. No endogenous interferences were found either in blank plasma pools or in any of the 11 healthy volunteers’ plasma specimens (Fig. 1a). Elution times of similar compounds

Conclusion

The proposed method proved to possess adequate specificity, sensitivity, accuracy and precision for a reliable determination of dThd and dUrd in plasma of patients with symptoms suggestive of MNGIE, confirming the drastic reduction of the enzymatic activity found in buffy coats of patients [4], [5], [6] and is validated according to recommended guidelines [10]. The minimal sample pretreatment allows patient samples to be processed in a short time. Moreover, the simple reversed-phase HPLC-UV

Acknowledgments

The skillful technical assistance of Carmina Candela, Anita Calisti and Monica Balboni is gratefully acknowledged. Cecilia Baroncini edited the English text.

We also thank Rita Rinaldi and Loris Pironi from St. Orsola-Malpighi Hospital in Bologna (Italy) and Costanza Lamperti and Massimo Zeviani from National Neurological Institute Carlo Besta in Milan (Italy) for referring the samples. We are also grateful to the MNGIE patients and their families for participating to this study. This project

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