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. 2026 Jul 31;40(9):e70583. doi: 10.1002/bmc.70583

Development and Validation of Simultaneous Quantification of 6TGN and 6MMPN in Whole Blood Using LC–MS/MS: An Application to Therapeutic Drug Monitoring in Hematological Malignancies and in Inflammatory Bowel Diseases

Marie Allard 1, Nicolas Vignal 1, Aude Loiseau 1, Hélène Sauvageon 1, Yves Médard 1, Alain Plé 1, Evelyne Jacqz‐Aigrain 1, Samia Mourah 1,2, Lauriane Goldwirt 1,2,✉
PMCID: PMC13428185  PMID: 42538758

ABSTRACT

Thiopurine immunosuppressive drugs are indicated in children with acute lymphoblastic leukemia and in both children and adults with chronic inflammatory diseases. Therapeutic drug monitoring (TDM) of its DNA‐incorporated metabolites 6‐thioguanine nucleotides (6TGN) and 6‐methylmercaptopurine nucleotides (6MMPN) is of high interest to optimize efficacy and prevent toxicity. The objective of this study was to develop and validate a sensitive and simple LC–MS/MS method for the simultaneous quantification of 6TGN and 6MMPN in whole blood for TDM. Following red blood cell count and whole blood extraction with dithiothreitol plus acidic hydrolysis at 100°C, quantification was performed using an LC–MS/MS in positive ionization mode. Calibration curves were linear over the range of 20–1000 ng/mL for 6TGN and 20–20,000 ng/mL for 6MMPN, with correlation coefficients above 0.99 for all analytes. Intra‐ and inter‐day precisions were below 9.45%. Extraction recovery and matrix effects reached 78.9%–83.2% and 121% for 6TGN and 92.2%–95.2% and 112%–118% for 6MMPN, respectively. The method was applied to 749 clinical samples from patients with acute lymphoblastic leukemia or chronic inflammatory diseases. A sensitive and simple LC–MS/MS method was validated for 6TGN and 6MMPN in whole blood and was successfully applied to clinical samples for TDM.

Keywords: 6MMPN, 6TGN, therapeutic drug monitoring, thiopurine, whole blood

1. Introduction

Thiopurine immunosuppressive drugs azathioprine (AZA), 6‐mercaptopurine (6‐MP), and thioguanine are indicated in children with acute lymphoblastic leukemia (ALL) (Bell et al. 2004; Kamps et al. 2010; Schmiegelow et al. 2010) and in both children and adults with chronic inflammatory diseases (Al Hadithy et al. 2005; Derijks et al. 2004, 2006; Movva et al. 2017). In ALL, thiopurine induction and consolidation treatments, up to 2–3 years, improve the chances of long‐term remission (Rivera et al. 1993; Schmiegelow et al. 2014). In Crohn's disease, AZA and 6‐MP therapies are associated with clinical response, especially with treatment duration above 17 weeks (Prefontaine et al. 2009). In ulcerative colitis, AZA treatment is correlated to an absolute risk reduction of relapse of 23% (Gisbert et al. 2009). Thiopurines drugs have an immunosuppressive and antiproliferative role by incorporating their metabolites into DNA, which inhibit de novo synthesis of purine bases. Furthermore, 6TGN‐triphosphate metabolite inhibits RAC‐1 activity involved in T cell maturation and proliferation, leading to T lymphocyte apoptosis (Dervieux et al. 2001; Larsen et al. 2021; Seinen et al. 2016).

In the presence of glutathione‐S‐transferase, the prodrug AZA reacts with reduced glutathione to form 6‐MP (Bostrom and Erdmann 1993; Stocco et al. 2014). Before being active, 6‐MP is metabolized into 6‐thioguanine nucleotides (6TGN) and 6‐methylmercaptopurine nucleotides (6MMPN) (Van Scoik et al. 1985). 6‐MP metabolism is affected by multiple pharmacogenetic interactions of thiopurine methyltransferase (Abaji and Krajinovic 2017), inosine triphosphate pyrophosphatase (Stocco et al. 2009) and nudix hydroxylase 15 (Cuffari et al. 1996). Furthermore, high interindividual variability in thiopurines metabolism in both adults and children has been widely described (Adam de Beaumais et al. 2022). Indeed, both efficacy and side effects outcomes are influenced by exposure to metabolites and pharmacogenetic polymorphisms of NUDT15, ITPA, and TMPT. Elevated 6TGN concentrations can lead to myelosuppression, whereas elevated 6MMPN concentrations are associated with hepatotoxicity occurrence (Dubinsky et al. 2000). Moreover, extremely high 6MMPN levels can also cause myelosuppression (Adam de Beaumais et al. 2011; Boulieu et al. 1997). These various factors of variability, as well as the need to prevent toxicity and optimize efficacy, support the need for therapeutic drug monitoring (TDM). Currently, red blood cell (RBC) concentrations of metabolites are used as a reflection of target tissues concentrations. In Crohn's disease, the target 6TGN concentration to achieve efficacy is 235 pmol/8 × 108 RBC (Dubinsky et al. 2000; Feng et al. 2019), and toxicity can occur above 450 pmol/8 × 108 RBC (Dubinsky et al. 2000). Regarding 6MMPN, only maximum concentrations have been described, and they should not exceed 5700 pmol/8 × 108 RBC to prevent adverse effects (Dubinsky et al. 2000).

Historically, TDM of thiopurine drugs was performed on washed RBC using HPLC coupled with UV detection (Adam de Beaumais et al. 2022; Boulieu and Dervieux 1999; Dervieux and Boulieu 1998) and on whole blood using LC–MS/MS (Kirchherr et al. 2013; Miao et al. 2020; Yoo et al. 2018; Zochowska et al. 2016). The washed RBC approach requires a time‐consuming double washing step that is a potential source of pre‐analytical variability, furthermore no difference was found between 6TGN and 6MMPN in blood and in washed RBC when normalized to 8 × 108 RBC (Pike et al. 2001). Earlier LC–MS/MS methods used non‐specific internal standards (IS) such as 8‐bromoadenine (Dervieux et al. 2005) or bisoprolol (Rosdiana et al. 2021), which do not fully account for the hydrolysis‐dependent derivatization of 6MMPN during sample preparation; the use of stable isotope‐labelled IS (SIL‐IS) matching each analyte overcomes this limitation.

The objective of this study was to develop and validate a sensitive and simple LC–MS/MS method for the simultaneous quantification of 6TGN and 6MMPN in whole blood and to apply this method to clinical samples for TDM.

2. Material and Methods

2.1. Chemicals and Reagents

6‐Thioguanine (6TGN, MW = 167.2 g·mol−1), 6‐methylmercaptopurine (6MMPN, MW = 166.2 g·mol−1), and the IS [13C3,15N]‐6TGN and [13C,2H3]‐6MMPN were obtained from Alsachim (Illkirch, France). Chemical structures are presented in Figure 1. Formic acid was obtained from Honeywell Fluka (Charlotte, USA), dithiothreitol (DTT) and perchloric acid (HClO4) were obtained from Sigma Aldrich (Saint Louis, USA) and dimethyl sulfoxide (DMSO) from Carlo Erba (Val‐de‐Reuil, France). LCMS‐grade methanol was obtained from Merck (Darmstadt, Germany). Purified water was obtained from a water purification system Milli‐Q Integral 3 by Millipore Sigma (Burlington, USA). Non‐therapeutic human blood was obtained from l'Etablissement Français du Sang (Rungis, France, agreement #4600003990).

FIGURE 1.

FIGURE 1

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Chemical structures of 6TGN and 6MMPN. Red arrows show how precursors are fragmented into quantification products for 6TGN (m/z 167.992 ➔ 151.042) and for 6MMPN(d) (m/z 158.011 ➔ 110.0), the aminocarbonyl‐imidazole derivative formed during acidic hydrolysis.

2.2. Equipment and LC–MS/MS Conditions

Liquid chromatography consisted of a Vanquish Flex Binary. Separation was performed on an Acquity UPLC BEH C18 (2.1 × 50 mm; pore size 1.7 μm; Waters, Milford, MA, USA) analytical column housed in a thermostated compartment at 25°C. The chromatography system was coupled to a triple quadrupole TSQ Altis TSQ02‐10002 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), equipped with an electrospray interface and controlled by the TraceFinder Version 5.2.SP3 software.

The mobile phase used for chromatography was ultrapure water/formic acid (99.9:0.1, V/V) (Phase A) and methanol/formic acid (99.9:0.1, V/V) (Phase B). The gradient was as follows: 95:5 (A:B, v/v) from 0 to 1 min, linear gradient to 10:90 (A:B) from 1 to 1.5 min, isocratic hold at 10:90 until 2.5 min, re‐equilibration to 95:5 over 0.5 min, and final equilibration at 95:5 until 4 min. The mobile phase flow rate was 300 μL/min. The column temperature was maintained at 25°C and the autosampler at 10°C.

MS conditions were determined as follows: samples were analyzed using an electrospray probe operating in positive ionization mode. Ion transfer tube temperature and vaporizer temperature were set at 325°C and 350°C, respectively, with sheath gas at 50 Arb, Aux gas at 10 Arb, and Sweep gas at 1 Arb. Capillary voltage was set at 3.5 kV. Argon was used as collision gas with a flow of 1.5 mTorr.

Determination of optimal settings and MS/MS transitions was obtained by direct infusion into the MS/MS detector of individual or combined solutions of each drug at 1 ng/mL in methanol. Two transitions were selected for each analyte to improve specificity: one was used for quantitation and one for confirmation. Production of the amino‐carbonyl imidazole derivate (6MMPN(d)) is achieved by acidic hydrolysis at high temperature of a 6MMPN working solution and was used for MS optimization. The monitored m/z values and optimized parameters for the precursor and product ions are summarized in Table 1.

TABLE 1.

LC–MS/MS parameters for the analysis of 6TGN, 6MMPN, and internal standards.

Analyte Polarity Retention time (min) RF lens (V) Precursor (m/z) Product (m/z) quantification Quantification collision energy (V) Product (m/z) confirmation Collision energy (V) confirmation
6MMPN(d) Positive 1.9 27 158.011 110.00 12.00 55.07 31
[13C‐2H3]‐6MMPN(d) Positive 1.9 27 162.000 110.02 24.92 55.06 24.92
6TGN Positive 0.9 27 167.992 151.042 19.95 134.042 21.98
[13C3‐15N]‐6TGN Positive 0.9 27 172.025 155.042 20 138.071 21.76
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2.3. Preparation of Reagents, Calibrators and Quality Control (QC) Samples

Stock solutions (1 mg/mL) of 6TGN and [13C3,15N]‐6TGN were prepared in DMSO, and stock solutions of 6MMP and [13C,2H3]‐6MMP were prepared in methanol according to manufacturer's recommendations.

Working solutions of 6TGN and 6MMPN at 1, 10, and 100 μg/mL were prepared by serial dilution of stock solutions into blank human whole blood. These working solutions were further diluted into blank whole blood to generate the final calibrator concentrations: 20, 50, 100, 250, 500, 750, and 1000 ng/mL for 6TGN and 20, 100, 1000, 2500, 5000, 10,000, and 20,000 ng/mL for 6MMPN. Three QCs were prepared for 6TGN (40, 500, and 700 ng/mL) and 6MMPN (500, 2000, and 7500 ng/mL). IS solutions containing [13C3,15N]‐6TGN and [13C,2H3]‐6MMPN at 1000 ng/mL were prepared in purified water. DTT was diluted in water at 1 M for stock solutions and at 0.2 M for working solutions. Stock and working solutions, calibrators, and QCs were stored at −80°C and non‐therapeutic human blood at −20°C.

2.4. Sample Extraction Method

On the day of procedure, calibrators, QCs, and heparinized whole blood samples from patients treated with thiopurine were left thawing at room temperature. In Eppendorf tubes, 50 μL of sample/calibrant/QC were mixed with 30 μL of DTT (0.2 M), 50 μL of IS (1000 ng/mL), and 320 μL of water. Fifty microliters of HClO4 (70% V/V) were then added before vigorous mixing for 1 min.

After 14,000 rpm centrifugation for 10 min, supernatant was transferred into a 96‐well plate and left for 1 h at 100°C. This acidic hydrolysis at high temperature serves to release the DNA‐incorporated metabolites and to convert 6MMPN into 6MMPN(d) that can be quantified by tandem mass spectrometry.

After cooling to room temperature, the plate was centrifuged at 3000 rpm for 5 min and 2 μL were injected for LC–MS/MS analysis.

2.5. Validation Procedure

Analytical methodology was validated according to the guidelines outlined in the International Council for Harmonization (ICH) (Committee for Medicinal Products for Human Use 2022). As part of validation, linearity, accuracy, precision, recovery, matrix effect, sensitivity, selectivity, and stability were assessed.

2.5.1. Linearity

Quantification was performed using the ratio of integrated peak areas of 6TGN or 6MMPN over corresponding IS. Calibration curve linearity was assessed by linear regression analysis. Calibration curves were obtained with a weighting factor of 1/X and were assessed by linear regression analysis for 6TGN and second order quadratic regression for 6MMPN.

2.5.2. Precision and Accuracy

Within‐ and between‐run accuracy (measured concentration/nominal concentration × 100) and precision ((standard deviation/mean) × 100) (CV%) were determined for QCs (n = 30, for the three levels). CV% for the estimated concentrations and bias should not exceed ±15%.

2.5.3. Sensitivity and Selectivity

The lower limit of quantification (LLOQ) was defined as the concentration of the lowest tested calibrator with an accuracy between 80% and 120% (n = 6) and a precision, expressed as the CV% within 20%.

The upper limit of quantification (ULOQ) was defined as the concentration of the highest calibrator with accuracy between 85% and 115% (n = 6) and a precision, expressed as the CV% within 15%.

Selectivity was assessed by analyzing blank blood samples from non‐treated patients compared with 6TGN and 6MMPN spiked samples at the retention times of the analytes and IS.

2.5.4. Matrix Effect and Extraction Recovery

Matrix effect was assessed by the ratio of the analyte peak areas of extracted blank samples spiked with stock solutions over analyte peak areas of H2O/MeOH 95:5 (V/V) solutions at identical concentrations according to EMA Guidelines (Committee for Medicinal Products for Human Use 2022). Extraction recoveries were evaluated by analyzing the ratio between peak areas of extracted over non‐extracted QC samples.

2.5.5. Carry‐Over and Dilution

Carry‐over was evaluated by injecting three high concentration samples (1000 and 20,000 ng/mL for 6TGN and 6MMPN, respectively) followed by three low concentration samples (20 ng/mL for both compounds); this was repeated five times.

Five out‐of‐range concentration samples were prepared and then diluted (1:2 and 1:5) in blank matrix. Samples were analyzed pure and diluted; accuracy and precision should be within ±15%.

2.5.6. Stability

Stability of 6TGN and 6MMPN in whole blood was assessed based on published data from de Graaf et al. (2010), who demonstrated stability for at least 7 days at −20°C and across three freeze–thaw cycles. In the present study, samples were immediately frozen at −80°C upon collection and analyzed within established stability windows. Bench‐top stability (4 h at room temperature) and autosampler stability (24 h at 10°C) of processed samples were verified during method development and remained within ±10% of nominal concentrations.

2.5.7. External QCs

External QCs provided by SKML (Nijmegen, Netherlands) were regularly analyzed using the process previously described.

2.6. Clinical Application

This new LC–MS/MS quantification method was successfully applied to patients treated with thiopurine for ALL or for chronic inflammatory diseases. Data were collected from medical records and laboratory software and an institutional review board exemption was obtained to perform retrospective chart review of patients in accordance with French legislation. 6TGN and 6MMPN exposure comparison according to clinical indication were performed using unpaired t‐test (GraphPad Prism Version 10.4.2 for Windows, GraphPad Software, Boston, Massachusetts USA). Blood samples were collected in heparin‐containing tubes before drug intake (trough concentration) at steady‐state after at least 4 weeks of daily dosing. Samples were processed as follows: 500 μL whole blood was immediately frozen and kept at −80°C until analysis according to stability data previously published (de Graaf et al. 2010), 1000 μL were used to quantify RBC count using an automatic hematology analyzer XN‐9000 (Sysmex, France). Therefore, 6TGN and 6MMPN concentrations were expressed in pmoles per 8 × 108 RBC as follows: 6TGN or 6MMPN (mol/8 × 108 RBC) = [6TGN or 6MMPN (pg/μL)] × 800 / ([RBC count per mL] × MW). For analysis, samples were thawed and processed as described in Section 2.4.

3. Results

3.1. LC–MS/MS Conditions

Chromatograms of blank plasma, LLOQ, and the patient's sample are presented in Figure 2. The retention time of each drug and its stable isotope used as an IS was consistent per analyte. Chromatograms showed sharp, symmetrical peaks, and no interference was highlighted in extracted blank whole blood. This assay is characterized by a fast sample workup combined with short retention times (0.9–1.9 min). The chromatographic run time of 4 min with one tray of gradient 5:95 improved the resolution of signals and column equilibration.

FIGURE 2.

FIGURE 2

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Chromatograms of 6TGN, 6MMPN, and internal standards. (a) 6TGN blank sample; (b) 6MMPN blank sample; (c) 6TGN LLOQ (20 ng/mL); (d) 6MMPN LLOQ (20 ng/mL); (e) 6TGN patient sample (237 ng/mL); (f) 6MMPN patient sample (451 ng/mL); (g) 6TGN internal standard [13C3,15N]‐6TGN; (h) 6MMPN internal standard [13C,2H3]‐6MMP.

3.2. Linearity

For 6TGN, regression correlation coefficients were all > 0.990, and regression comparison showed similar slopes (p = 0.30), with a pooled slope of 1.019 and similar Y‐intercepts (p > 0.9999) with a pooled intercept of −7.227, giving a mean calibration curve of Y = 1.019X − 7.416 (R 2 = 0.999).

For 6MMPN, regression correlation coefficients were all > 0.999 and regression comparison showed similar slopes (p > 0.9999), with a pooled slope of 1.000 and similar Y‐intercepts (p > 0.9999) with a pooled intercept of −1.352, giving a mean calibration curve of Y = 1.000X − 1.352 (R 2 = 0.999) (Figure 3).

FIGURE 3.

FIGURE 3

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6TGN and 6MMP standard curve.

3.3. Precision and Accuracy

Precision and accuracy determined across all QC levels as presented in Table 2 displayed intra‐assay precision below −3.51% and inter‐assay precision below 9.45%, which are consistent with ICH recommendations (Committee for Medicinal Products for Human Use 2022).

TABLE 2.

Data detailing intra‐assay (n = 30) and inter‐assay (n = 30) accuracy and precision and LLOQ mean accuracy of 6TGN and 6MMPN.

Nominal (ng/mL) 6TGN
Intra‐assay Inter‐assay
%RE %CV %RE %CV
40 −9.11 3.51 −13.52 9.45
400 3.18 1.71 2.02 6.24
700 11.34 2.24 6.36 6.40
LLOQ (20 ng/mL) % mean accuracy
−4.08
Nominal (ng/mL) 6MMPN
Intra‐assay Inter‐assay
%RE %CV %RE %CV
500 8.73 3.15 4.61 9.22
2000 11.66 1.40 5.57 7.15
7500 3.82 1.50 4.41 6.92
LLOQ (20 ng/mL) % mean accuracy
1.54
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3.4. Sensitivity and Selectivity

The LLOQ was validated at 20 ng/mL for 6TGN and 6MMPN, with mean accuracies of −4.08% for 6TGN and 1.54% for 6MMPN (Table 2). For both analytes, no peak was observed in the tested blank whole blood samples, thus ensuring the selectivity of the method.

3.5. Extraction Recovery and Matrix Effects

For 6TGN, recovery ranged from 78.88% to 83.20%, whereas matrix effects were calculated at 121% for both QC levels. For 6MMPN, recoveries were higher, at 92.20% for lower QC and 95.19% for higher QC, whereas matrix effects were found at 112% and 118%, respectively (Table 3).

TABLE 3.

Extraction recovery and matrix effect.

6TGN
Extraction recovery Matrix effect
Low Q.C.
Mean value 78.9% 121%
Relative standard deviation 4.7% 6.7%
High Q.C.
Mean value 83.2% 121%
Relative standard deviation 5.3% 3.8%
Contamination rate 0.062%
1:2 dilution (mean bias) −9.7%
1:5 dilution (mean bias) −10.3%
6MMPN
Extraction recovery Matrix effect
Low Q.C.
Mean value 92.2% 112%
Relative standard deviation 11.9% 13.7%
High Q.C.
Mean value 95.2% 118%
Relative standard deviation 11.8% 13.6%
Contamination rate 0.009%
1:2 dilution (mean bias) −0.8%
1:5 dilution (mean bias) 9.2%
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3.6. Carry‐Over and Dilution

Contamination rates were 0.062% and 0.009% for 6TGN and 6MMPN, respectively. Thus, no carry‐over was observed until 1000 ng/mL for 6TGN and 20,000 ng/mL for 6MMPN. All 1:2 and 1:5 dilutions differed from less than 11% for both compounds (Table 3).

3.7. External QCs

Nine external QC campaigns were available with two samples per campaign. Depending on the campaign, 10–12 laboratories participated in our peer group. 6TGN concentrations ranged from 1.0 to 4.0 μmol/L (167.2–668.8 ng/mL) and 6MMPN from 2.9 to 30.7 μmol/L (482.0–5102.3 ng/mL) in 2024, 2025, and early 2026 peer group. Trueness was −9.4% and −4.9% for 6TGN and 6MMPN, respectively, in 2024 and −13% and −10% for 6TGN and 6MMPN, respectively, in 2025. The detailed results are presented in Table 4.

TABLE 4.

External quality control results.

6TGN 6MMPN
Peer group results (μmol/L) Peer group results (ng/mL) Laboratory results (ng/mL) Bias (%) Peer group results (μmol/L) Peer group results (ng/mL) Laboratory results (ng/mL) Bias (%)
2024‐1A 2.51 419.7 434.72 3.59% 7.90 1313.0 1396.08 6.33%
2024‐1B 2.99 499.9 518.32 3.68% 17.80 2958.4 2958.36 0.00%
2024‐2A 1.45 242.4 250.8 3.45% 25.90 4304.6 4454.16 3.47%
2024‐2B 2.95 493.2 518.32 5.08% 9.20 1529.0 1662 8.70%
2024‐3A 1.50 250.8 284.24 13.33% 23.40 3889.1 4071.9 4.70%
2024‐3B 2.40 401.3 443.08 10.42% 12.00 1994.4 2060.88 3.33%
2024‐4A 3.00 501.6 518.32 3.33% 10.00 1662.0 1728.48 4.00%
2024‐4B 4.00 668.8 702.24 5.00% 4.80 797.8 764.52 −4.17%
2025‐1A 1.90 317.7 367.84 15.79% 25.20 4188.2 4470.78 6.75%
2025‐1B 2.55 426.4 468.16 9.80% 6.30 1047.1 1096.92 4.76%
2025‐2A 2.00 334.4 351.12 5.00% 16.60 2758.9 2925.12 6.02%
2025‐2B 2.80 468.2 484.88 3.57% 4.80 797.8 797.76 0.00%
2025‐3A 2.05 342.8 367.84 7.32% 11.70 1944.5 2060.88 5.98%
2025‐3B 3.16 528.4 535.04 1.27% 3.60 598.3 648.18 8.33%
2025‐4A 1.00 167.2 183.92 10.00% 18.00 2991.6 2991.6 0.00%
2025‐4B 3.25 543.4 601.92 10.77% 2.90 482.0 465.36 −3.45%
2026‐1A 2.50 418.0 476.52 14.00% 30.70 5102.3 5135.58 0.65%
2026‐1B 1.60 267.5 294.272 10.00% 11.90 1977.8 1927.92 −2.52%
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3.8. Clinical Application

TDM of 6TGN and 6MMPN was successfully applied to 749 patients between January 2025 and June 2025. In this population consisting of adults and children with ALL or chronic inflammatory diseases, concentrations presented in Table 5 ranged from below 20 to 1288 ng/mL for 6TGN (< 20 and 2282 pmol/8 × 108 RBC) and < 20 and 32,868 ng/mL for 6MMPN (< 20 and 41,417 pmol/8 × 108 RBC). Concentrations stratified by pathology, co‐medication, and age are presented in Table 5, including 656 patients after exclusion of 93 patients treated for alternative autoimmune indications. Statistical results from the t‐test conducted on this cohort showed no significant differences in 6TGN and 6MMPN concentrations between IBD and autoimmune hepatitis patients (p = 0.35 and p = 0.46, respectively). However, 6TGN and 6MMPN concentrations in acute leukemia show significant differences with IBD and autoimmune hepatitis (p < 0.0001).

TABLE 5.

6TGN and 6MMPN concentrations measured in 749 samples presented according to pathology, main co‐medications and age. Results are presented as N (%) and median [IQR].

Crohn's disease Ulcerative colitis Autoimmune hepatitis Acute leukemia
Patients' distribution 286 (38%) 135 (18%) 108 (14%) 127 (17%)
Male 152 (53%) 69 (51%) 32 (30%) 68 (54%)
Dose/kg 1.71 [1.35–2.08] 1.72 [1.30–2.04] 1.44 [0.96–1.86] 1.74 [1.21–2.27]
6TGN concentrations (pmol/8*108 RBC) 213 [128–288] 276 [184–406] 211 [132–337] 358 [271–557]
6MMPN concentrations (pmol/8*108 RBC) 680 [267–1543] 489 [178–1365] 402 [205–843] 6572 [2668–16,080]
Co‐medications Anti‐TNF Anti‐TNF free Anti‐TNF Anti‐TNF free Corticosteroids Corticosteroid‐free Methotrexate Methotrexate‐free
Patients' distribution 190 (66%) 96 (34%) 60 (44%) 75 (56%) 35 (32%) 73 (68%) 100 (79%) 27 (21%)
Thiopurine dose (mg/kg) 1.73 [1.40–2.08] 1.69 [1.20–2.09] 1.68 [1.42–2.05] 1.74 [1.30–2.03] 1.66 [1.13–1.83] 1.37 [0.94–1.89] 1.69 [1.23–2.22] 1.93 [1.14–2.35]
6TGN concentrations (pmol/8*108 RBC) 212 [120–290] 220 [140–287] 226 [162–331] 308 [196–428] 179 [90–336] 224 [162–345] 363 [274–578] 309 [250–474]
6TGN concentration below LLOQ 29 (15%) 3 (3%) 7 (12%) 2 (3%) 0 (0%) 3 (4%) 3 (3%) 1 (4%)
6MMPN concentrations (pmol/8*108 RBC) 732 [303–1548] 619 [254–1512] 461 [200–1364] 507 [172–1347] 329 [144–697] 537 [247–1535] 6743 [2708–14,814] 5849 [2314–19,650]
6MMPN concentration below LLOQ 30 (16%) 2 (2%) 6 (10%) 2 (3%) 0 (0%) 9 (12%) 2 (2%) 2 (7%)
Age < 14 years old ≥ 14 years old < 14 years old ≥ 14 years old < 14 years old ≥ 14 years old < 14 years old ≥ 14 years old
Patients' distribution 61 (21%) 225 (79%) 38 (28%) 97 (72%) 19 (18%) 89 (82%) 110 (87%) 17 (13%)
Thiopurine dose (mg/kg) 1.70 [1.47–2.13] 1.72 [1.32–2.08] 1.74 [1.36–2.07] 1.72 [1.30–2.02] 1.79 [0.86–1.86] 1.37 [0.96–1.85] 1.80 [1.30–2.29] 1.25 [1.04–1.92]
6TGN concentrations (pmol/8*108 RBC) 192 [105–265] 219 [138–295] 284 [196–408] 253 [183–371] 237 [149–380] 208 [132–320] 360 [273–569] 336 [266–423]
6TGN concentration below LLOQ 2 (3%) 30 (13%) 1 (3%) 8 (8%) 1 (5%) 2 (2%) 2 (2%) 2 (12%)
6MMPN concentrations (pmol/8*108 RBC) 662 [355–1389] 713 [261–1565] 649 [300–1720] 417 [161–1260] 615 [182–767] 397 [213–855] 6728 [2668–15,564] 5682 [2523–17,741]
6MMPN concentration below LLOQ 2 (3%) 30 (13%) 1 (3%) 7 (7%) 1 (5%) 8 (9%) 3 (3%) 1 (6%)
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4. Discussion and Conclusion

TDM of thiopurine metabolites requires highly sensitive, specific measurement and a wider linearity range than previously published methods (Miao et al. 2020). In this context, we developed and validated an LC–MS/MS method for thiopurine drug metabolites measurements according to EMA guidelines (Committee for Medicinal Products for Human Use 2022) and successfully applied it to patients regardless of their pathology.

In a 4‐min runtime, we successfully separated 6TGN and 6MMPN along with their respective IS. Considering that 6MMPN and [13C,2H3]‐6MMPN undergo hydrolysis during the extraction step, the use of stable labelled isotopes as IS improved the specificity compared with techniques using bisoprolol (Rosdiana et al. 2021) or 8‐bromoadenine (Dervieux et al. 2005) as IS. This method required a lower sample volume to reach a higher sensitivity (Hofmann et al. 2012; Miao et al. 2020; Zochowska et al. 2016), probably due to organic solvent phase choice: indeed, we achieved a better sensibility for 6TGN with the use of methanol instead of acetonitrile as organic mobile phase. Our method linearity range was appropriate for the concentrations found in our patients' cohort, as 94% of the samples were above LLOQ. Compared with previously published methods, we drastically simplified the preanalytical procedure by suppressing double washing RBC steps (Adam de Beaumais et al. 2022; Chu et al. 2026; Dervieux et al. 2005; Kirchherr et al. 2013; Mei et al. 2017; Rosdiana et al. 2021; Yoo et al. 2018). Such process has already been described and showed to shorten sample preparation without altering quantifying quality (Hofmann et al. 2012; Miao et al. 2020; Zochowska et al. 2016). These methods all showed good sensitivity and specificity, and focused on maintaining same therapeutic target expressed with RBC normalization, although expressing results according to hemoglobin concentration has also been proposed without clinical validation of a new therapeutic target (Kirchherr et al. 2013).

Interindividual variability was very high for 6MMPN and 6TGN to a lower extent, likely due to metabolism polymorphism (Tamm et al. 2017) which confirms the interest of TDM in patients treated with thiopurine.

No difference between 6TGN exposure in bowel inflammatory disease patients was observed according to their age nor co‐medications; nevertheless, 6TGN and 6MMPN were markedly higher in patients treated for acute leukemia, which is likely attributable to more intensive thiopurine dosing protocols and different pharmacogenetic selection pressures in this population. This observation raises the question of optimizing 6TGN and 6MMPN TDM interpretations according to pathology and treatment duration, as the currently accepted therapeutic targets were established in IBD cohorts using washed RBC methods and may not be directly transposable to the ALL setting.

In conclusion, we have developed and validated a simple and rapid run‐time assay for the quantification of 6TGN and 6MMPN using LC–MS/MS. This assay is of high interest in TDM of patients with ALL or chronic inflammatory diseases and could be a useful tool for individual treatment optimization.

Author Contributions

Conceptualization: Lauriane Goldwirt; Data curation: Marie Allard, Nicolas Vignal, Alain Ple; Formal analysis, Methodology, Sofware, Validation, Vizualization and Writing original draft: Marie Allard, Nicolas Vignal, Lauriane Goldwirt; Resources: Marie Allard, Nicolas Vignal, Alain Ple, Hélène Sauvageon, Yves Médard, Aude Loiseau, Evelyne Jacqz‐Aigrain, Samia Mourah Lauriane Goldwirt; Supervision: Lauriane Goldwirt; Writing review and editing: Lauriane Goldwirt.

Funding

The authors have nothing to report.

Conflicts of Interest

L.G. declares honoraria for the advisory board for AbbVie, Ipsen, Promise, Takeda, Serb, as well as travel, accommodation, and expenses from Novartis. S.M. declares honoraria for the advisory board for Pierre Fabre, Novartis, and Biocartis. The other co‐authors declare no conflicts of interest.

Acknowledgments

We thank Patricia Maslanka and Aïcha Laghzal for their technical contribution.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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