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Monitoring of thiopurine metabolites - a high-performance liquid chromatography method for clinical use.

Journal of pharmaceutical and biomedical analysis (2012-12-25)
Svante Vikingsson, Sven Almer, Curt Peterson, Björn Carlsson, Martin Josefsson
ABSTRACT

A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials. Ten patient samples were analysed to demonstrate clinical application and to establish pilot ranges for all analytes. The method measured thioguanosine mono-(TGMP), di-(TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine). LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8 × 10⁸ RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 4 °C for 8 h after sampling.

MATERIALS
Product Number
Brand
Product Description

Supelco
Tetrabutylammonium phosphate monobasic solution, suitable for ion pair chromatography, LiChropur, concentrate, ampule
Sigma-Aldrich
Tetrabutylammonium hydroxide solution, technical, ~40% in H2O (~1.5 M)
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Tetrabutylammonium perchlorate, ≥95.0% (T)
Supelco
Tetrabutylammonium hydroxide solution, ~40% in water, suitable for ion chromatography
Sigma-Aldrich
Tetrabutylammonium phosphate monobasic solution, 1.0 M in H2O
Sigma-Aldrich
Tetrabutylammonium cyanide, 95%
Sigma-Aldrich
Tetrabutylammonium cyanide, technical, ≥80%
Supelco
Tetrabutylammonium perchlorate, for electrochemical analysis, ≥99.0%
Sigma-Aldrich
Tetrabutylammonium hydroxide solution, 1.0 M in methanol
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Tetrabutylammonium hydroxide solution, 40 wt. % in H2O
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Tetrabutylammonium hydrogensulfate, 97%
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6-Mercaptopurine monohydrate, 98%
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Tetrabutylammonium hydroxide solution, 54.0-56.0% in H2O
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Potassium permanganate, ACS reagent, ≥99.0%
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Potassium permanganate, ≤150 μm particle size, 97%
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Potassium permanganate, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99%
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Potassium permanganate, ACS reagent, ≥99.0%, low in mercury
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Potassium permanganate, puriss. p.a., ACS reagent, Hg ≤0.000005%, ≥99.0% (RT)
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6-Thioguanine, ≥98%
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6-Thioguanine, Hybri-Max, 50 ×, γ-irradiated, lyophilized powder, BioXtra, suitable for hybridoma
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Tetrabutylammonium bromide solution, 50 wt. % in H2O
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Tetrabutylammonium iodide, reagent grade, 98%
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Tetrabutylammonium fluoride solution, 1.0 M in THF
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Tetrabutylammonium bromide, ACS reagent, ≥98.0%
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Tetrabutylammonium fluoride solution, 75 wt. % in H2O
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Tetrabutylammonium nitrate, 97%
Supelco
Tetrabutylammonium iodide, suitable for ion pair chromatography, LiChropur, ≥99.0%
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Tetrabutylammonium bisulfate solution, suitable for ion pair chromatography, LiChropur, concentrate, ampule
Sigma-Aldrich
Tetrabutylammonium phosphate monobasic, puriss., ≥99.0% (T)
Sigma-Aldrich
Tetrabutylammonium iodide, ≥99.0% (AT)