Analytical and Sample Preparation Protocol for Therapeutic Drug Monitoring of 12 Thiopurine Metabolites Related to Clinical Treatment of Inflammatory Bowel Disease

Abstract

Thiopurines (TP) represent an important therapeutic tool for the treatment of inflammatory bowel diseases (IBD) in the current situation of rising incidence and health care costs. The results of multiple clinical studies aimed at finding correlations between levels of TP metabolites and response of IBD patients to the treatment are, however, often controversial due to variability in analytical and sample preparation procedures among these studies. In this work, therefore, an updated analytical and sample preparation procedure for therapeutic drug monitoring (TDM) of TP metabolites in blood samples obtained from patients with IBD was proposed to establish a unified protocol. An advanced analytical method based on ion-exchange liquid chromatography hyphenated with tandem mass spectrometry (IEC-ESI-MS/MS) was used for the determination of the profiles of 12 individual TP metabolites in the particular steps of sample preparation procedure including blood collection, red blood cells (RBC) isolation, lysis, and storage. Favorable performance parameters of the IEC-ESI-MS/MS method (LLOQs 1⁻10 nmol/L, accuracy 95⁻105%, intra-day and inter-day precision < 10%, selectivity demonstrated via no sample matrix interferences) and acceptable stability (peak area fluctuations < 15%) of clinical samples under the proposed sample preparation conditions {(i) EDTA anticoagulant tube for the blood collection; (ii) 4 °C and 4 h between the sample collection and RBC isolation; (iii) phosphate-buffered saline for RBC washing and re-suspendation; (iv) -20 °C for RBC lysis and short-term storage; (v) 50 mmol/L phosphate buffer, pH 7.4, 10 mmol/L DTT as a stabilizing medium for TPN in RBC lysates} demonstrated the suitability of such protocol for a well-defined and reliable routine use in studies on thiopurines TDM.

Keywords: clinical analysis; comprehensive stability study; high performance liquid chromatography; inflammatory bowel diseases; mass spectrometry; profiling of thiopurines; sample treatment; therapeutic drug monitoring.

Figure 1

Scheme of thiopurine metabolism. AZA, azathioprine; 6-MMP, 6-methylmercaptopurine; TPMT, thiopurine S-methyltransferase; 6-MP, 6-mercaptopurine; XO, xanthine oxidase; 6-TU, 6-thiouric acid; HPRT, hypoxanthine guanine phosphoribosyltransferase; TIMP, 6-thioinosine 5′-monophosphate; TIDP, 6-thioinosine 5′-diphosphate; TITP, 6-thioinosine 5′-triphosphate; ITPA, inosine triphosphate pyrophosphatase; MeTIMP, 6-methylthioinosine 5′-monophosphate; MeTIDP, 6-methylthioinosine 5′-diphosphate; MeTITP, 6-methyl-thioinosine 5′-triphosphate; IMPHD, inosine monophosphate dehydrogenase; TXMP, 6-thioxanthine 5′-mono-phosphate; TGMP, 6-thioguanosine 5′-monophosphate; TGDP, 6-thioguanosine 5′-diphosphate; TGTP, 6-thio-guanosine 5′-triphosphate; 6-TG, 6-thioguanine; MeTGMP, 6-methylthioguanosine 5′-monophosphate; MeTGDP, 6-methylthioguanosine 5′-diphosphate; MeTGTP, 6-methylthioguanosine 5′-triphosphate.

Figure 2

Representative profile of 12 thiopurine nucleotides in clinical sample taken from IBD patient treated by AZA. The RBC lysate sample was prepared according to the Section 3.3 and Section 3.4 with optimized sample preparation parameters (summarized in Section 4). The IEC-ESI-QqQ method with optimized operating parameters was used for the analysis (Section 3.5 and Section 3.6).

Figure 3

Stability of thiopurine nucleotides in different stabilization media during 5-day storage at 4 °C. Media used: demineralized water, serving as a reference medium (left panels), 50 mmol/L EDTA (middle panels), 5 mmol/L phosphate buffer (right panels). IEC-ESI-QqQ method (Section 3.5 and Section 3.6) was used for the analysis. For the sample preparation see Section 3. Materials and Methods.

Figure 4

Influence of stabilization additive for phosphated thiopurines on detection signal. EIC chromatograms of MeTGMP; TGMP; MeTIMP and TIMP obtained with addition of EDTA and phosphate buffer (PB) as stabilizing agents. Standard solution of the same concentration was used for all analyses. Bottom panel: TIC chromatogram of the same sample. IEC-ESI-QqQ method (Section 3.5 and Section 3.6) was used for the analysis. For the sample preparation see Section 3. Materials and Methods.

Figure 5

Stability of thiopurine nucleotides in different anticoagulant syringes used for collection of blood samples and different holding times of such samples. Comparison of levels of methylthioguanosine and thioguanosine nucleotides (diagrams in upper half) and methylthioinosine nucleotides and thioinosine nucleotides (diagrams in lower half) in the samples collected into the EDTA or Li-Heparin syringes and hold for 4 h to the samples processed immediately (expressed as %). Dark blue line on the secondary axis represents levels of TPN expressed as pmol/8 × 10⁸ RBC in sample processed immediately.

Figure 6

Stability of thiopurine nucleotides in different RBC washing and dilution solutions. Comparison of levels of methylthioguanosine and thioguanosine nucleotides (diagrams in upper half) and methylthioinosine nucleotides and thioinosine nucleotides (diagrams in lower half) in the RBC samples washed and diluted with the saline or PBS and hold for 4 h to the samples processed immediately (expressed as %). Dark blue line on the secondary axis represents levels of TPN expressed as pmol/8 × 10⁸ RBC in sample processed immediately.

Figure 7

Stability of thiopurine nucleotides under different RBC lysis and short-term storage temperature. The ratios of TPN peak areas referring to the RBC lysed and stored for 24 h at −80 °C and −20 °C are expressed graphically as columns. The RBC lysates were prepared under optimum conditions: (i) EDTA anticoagulant tube for the blood collection; (ii) time between the sample collection and RBC isolation 0 h or 4 h at the temperature of 4 °C (upper and lower diagrams); (iii) phosphate-buffered saline for RBC washing (two-times) and re-suspendation (dilution 1:1); (iv) 50 mmol/L phosphate buffer with pH 7.4 and 10 mmol/L DTT additive as a stabilizing medium for TPN in RBC lysates.