An optimized UHPLC-MS/MS method for simultaneous determination of erythrocyte methotrexate and polyglutamylated metabolites: Application in pediatric patients with acute lymphoblastic leukemia

doi:10.21203/rs.3.rs-2598435/v1

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An optimized UHPLC-MS/MS method for simultaneous determination of erythrocyte methotrexate and polyglutamylated metabolites: Application in pediatric patients with acute lymphoblastic leukemia

https://doi.org/10.21203/rs.3.rs-2598435/v1

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Methotrexate (MTX), a widely used chemotherapeutic drug, is critical for achieving long-term complete remission in contemporary maintenance therapy of acute lymphoblastic leukemia (ALL). MTX is intracellularly converted into methotrexate polyglutamates (MTXPGn) by folylpolyglutamate synthase after transporting into the cells. The intracellular levels of active MTXPGn regulate the clinical efficacy and drug-related side effects, but it is still challenged by large inter-individual differences and narrow therapeutic index. Therefore, it is of great significance to develop a sensitive and stable method to monitor MTXPGs concentrations in the erythrocyte. To facilitate clinical application, an ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the quantification of MTX and MTXPGn in erythrocyte. Solid phase extraction was applied to sample clean-up followed by a gradient chromatographic separation. The elution consisting of methanol/water/2 mM ammonium acetate (flow rate: 0.4 mL/min). Linearity of the assay was assured in the range of 1-500 nM (R² > 0.99). The co-efficient of variation ranged from 2–11% for intraday precision and 0.4–8% for inter day precision. Recovery ranged from 62.51–99.75%, and the matrix-effect varied from 63.24–96.33%. Blood samples from 89 pediatric ALL patients were detected. The concentrations and distribution of MTXPGn in these samples were basically consistent with the current literatures, which confirmed that the optimized method for quantitative detection of MTXPGn in red blood cells had high sensitivity and accuracy. The sensitive method has been fully validated and successfully applied to determine the erythrocyte MTXPGn in pediatric ALL patients during continuation therapy.

Methotrexate

UHPLC-MS/MS

Methotrexate polyglutamates

childhood acute lymphoblastic leukemia

Methotrexate (MTX) is an anti-folate tumor drug with high clinical usage, which is mainly used in leukemia, lymphoma and rheumatic immune diseases. It exerts pharmacologic effects by inhibiting dihydrofolate reductase, disturbing the synthesis of thymidine and purine nucleotides, and finally blocking the synthesis of DNA in tumor cells. It has been reported that MTX enters blood cells and is rapidly transformed by leaf polyglutamate synthase (95% in 24h) to produce MTXPGn, a process attaching sequential γ-linked glutamic residues to MTX ^[1]. MTXPGn is not easy to penetrate cell membrane and exists in red blood cells (RBCs) for a long time. It is an active substance that mainly plays an anti-metabolic role which takes 4–8 weeks to reach steady state in RBCs and the elimination half-life is 1–4 weeks.

The cornerstone combination of weekly MTX (20 mg/m²/week) and daily mercaptopurines is critical for achieving long-term for contemporary maintenance therapy of ALL ^[2–3]. Considering the narrow therapeutic range of MTX ^[4] and the confounding factors such as gene polymorphisms and plasma protein levels ^[5], it is not surprising that, the pharmacokinetics/pharmacodynamics of MTX varied among individuals and even some patients discontinue MTX due to toxicity occurrence (hepatotoxicity and neutropenia) ^[6–7]. It is reported that the concentrations of intracellular metabolites are closely related to the clinical therapeutic effect ^[8–9], which is known to be related to the prognosis of pediatric acute lymphoblastic leukemia (ALL) patients ^[10–11]. Similarly, for the treatment of rheumatoid arthritis (RA), although MTX could bring the improved outcome with tolerable adverse reactions, the individual curative effect is different and difficult to predict ^[12]. Therapeutic drug monitoring (TDM) is one of the commonly used strategies to monitor the MTX clinical response profiles for patients and consequent adjustment of dosage regimes ^[13]. Therefore, it is imperative to recommend TDM for MTX and its active metabolites in pediatric ALL patients.

Development of a sufficiently sensitive method for simultaneous determination of MTX and its polyglutamylated metabolites in red blood cells would be necessary in therapeutic drug monitoring. Currently, several bioassays, such as immunoenzymatic assays^[14], high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) ^[15–17], have been applied in various clinical settings, among of which were limited to long analytical times, the interferences from analogues, low specificity and time consuming. Also, Boer et al. ^[17] measured the concentrations of MTXPG_1 − 5 by using stable isotope labeled internal standard, while they did not successfully detect MTXPG_6 − 7 in their research. Accordingly, this study aimed to establish a LC-MS/MS method for the detection of MTX and its six active metabolites in human erythrocyte lysate. This method has the advantages of high sensitivity, strong specificity and low detection limit. The mass spectrometry and liquid phase detection method are optimized to the best conditions, which is convenient for clinical laboratory application.

Materials

Methotrexate (4-amino-10-methylpterin glutamate, MTX), Methotrexate Diglutamate Trifluoroacetate (MTXPG₂), Methotrexate Triglutamate Trifluoroacetate (MTXPG₃), Methotrexate Tetraglutamate Trifluoroacetate (MTXPG₄), Methotrexate Hexaglutamate Trifluoroacetate (MTXPG₆), Methotrexate Heptaglutamate Trifluoroacetate (MTXPG₇), and internal standard 4-aminopterin glutamate (aminopterin; AO) were purchased from Toronto Research Chemicals (Toronto, Canada). Methotrexate Pentaglutamate Trifluoroacetate (MTXPG₅) was purchased from Schircks Laboratories (Jena, Switzerland). The chemical structures of MTX, MTXPG_2 − 7 and internal standard are shown in Fig. 1. Liquid chromatography-mass spectrometry (LC-MS) grade methanol and acetonitrile were obtained from Tedia (CT, USA), and ultra-pure water was prepared by Millipore Direct-Q water purification system (Millipore, MA, USA). Ammonium hydroxide, 25% ammonia water, and ammonium acetate were purchased from Sigma (Zwijndrecht, The Netherlands). Perchloric acid (HClO₄) was purchased from Guangzhou Chemical Reagent Factory. Blank human whole blood samples were obtained from volunteers of Sun Yat-sen Memorial Hospital of Sun Yat-sen University.

Lc-ms/ms Conditions

A Thermo UHPLC system (Thermo Fisher Scientific, Inc., MA, USA) coupled with a TSQ triple quadrupole spectrometer (Thermo Fisher Scientific, Inc) was applied for measurement. Detection was performed in selected reaction monitoring (SRM) mode. The data processing is carried out by using XCalibur 2.0 (Thermo Fisher Scientific) software package.

Chromatographic separation was performed at 40℃ using Waters Acquity UPLC BEH C₁₈ 1.7 µ m (2.1× 100 mm). The mobile phase was (A) 2 mM ammonium acetate and (B) Methanol with a flow rate of 0.3 mL/min. The pH for mobile phase A was adjusted to 10 with 25% ammonia water, The elution procedure was set as follows: at 0 min: 5%B, at 0–4 min, linear increase to 20%B, at 4–8 min increase to 36% B, at 8-8.5 min linear increase to 76% B, at 8.5-9.0 min drop to 5% B, and 9–13 min keep at 5% B.

MTXPGn and IS were ionized with electrospray ionization (ESI) in positive ion mode. The optimized mass spectrometry conditions were as follows: spray voltage: 3500 V, evaporation temperature: 350℃, sheath air pressure: 30psi, auxiliary air pressure: 10psi, temperature of ion transfer tube: 350℃. MTX and its six active metabolites (MTXPG_2 − 7) mainly generate [M + H]⁺ ion peak under ESI⁺ ionization mode, and generate the main fragment ions. Mass transition, fragment voltage and collision energy of MTXPGn and IS are shown in Table 1.

Table 1

The MS transitions of the precursors to the product ions in SRM mode.
Compound	Transitions(m/z)	Collision energy(eV)	Tune tube lens
MTX	455.3→308.3	19	123
MTX-d₃	458.6→311.2	20	177
MTX-PG2	584.6→308.3	25	70
MTX-PG3	713.7→308.3	20	96
MTX-PG4	842.5→308.3	38	105
MTX-PG5	971.5→308.3	58	136
MTX-PG6	1100.9→307.9	50	142
MTX-PG7	1229.9→307.6	57	165

Preparation Of Stock Solution And Working Solution

The standard stock solutions were prepared by dissolving separate weighted powder in 0.1 M ammonium hydroxide to achieving the target concentration of 100 µM for MTXPG_2 − 7, and 10 µM for the internal standard. Standard working solutions were prepared from further dilutions of the stock solution at the following concentrations:10, 20, 40, 80, 160, 320, 640, 1280, 2560 and 5000 nM. All the stock and working solutions were stored at a nominal temperature of -20℃ before use.

Preparation Of Erythrocyte Lysate

The EDTA anticoagulated whole blood refrigerated at 4℃ was gently overturned repeatedly and mixed evenly, then 1mL of whole blood was placed in a 2mL EP tube, centrifuged at 4000rpm for 10 min. The collected blood cells were washed gently with 1 mL sterile saline and centrifuge at 4000 rpm for 10 min. Repeat the washing and centrifugation steps two more times. After the third centrifugation, discard the supernatant, add 1mL of normal saline, gently blow and wash evenly, and make RBCs suspension. The lysis buffer of RBCs were obtained by diluting 300 µL of the RBCs resuspension solution with 600 µL of pure water followed by a 1-min vorterx. The lysis buffer was stored at -80℃ for testing.

Preparation Of Standard And Quality Control Samples

A batch of frozen blank RBCs were thawed by rotating at room temperature, and the final concentration of MTXPGn was 500 nM. A 10-point calibration curve was obtained by 1∶1 continuous dilution of blank RBCs. The concentrations of different metabolites MTXPGn were 1, 2, 4, 8, 16, 32, 64, 128, 256 and 500 nM respectively. Prepare a new calibration curve for each determination. The quality control (QC) samples are 1, 3, 200 and 400 nM, respectively. Samples were stored at − 80°C and thawed on ice.

Sample Pretreatment

A solid-phase extraction procedure was developed. Blood samples were treated to obtain erythrocyte lysate as described before. Four hundred microlitres of erythrocyte lysate mixed with 20 uL of internal standard solution, and deproteinization by 40 uL of perchloric acid. Precipitated proteins were removed by 2-min vortex and centrifugation for 10 min at 15000rpm at ambient temperature. After collection of the supernatants, 600uL of water was mixed with the 400 uL of supernatants. Then the analytes were extracted from the 900 uL of mixture by SPE using an Oasis HLB SPE column (Waters, Etten-Leur, The Netherlands) containing 30 mg adsorbent. The pre-activated SPE column was conditioned by washing the adsorbent with 1 mL of methanol followed by 2×1 mL of water. After absorption of the analytes and washing of 1mL of pure water, 200uL of methanol was used to elute the analytes. The collected elution was evaporated in a vacuum drying oven at 40℃. The residues was redissolved by 100 uL of mobile phase. The mixture was centrifuge at 15000 rpm for 10 min, and the supernatant was transferred to a sample bottle and place it in a 4℃ automatic sampler until analysis.

Methodological Verification

The bioassay was validated for specificity, lower limit of quantification (LLOQ), linearity, precision and accuracy, extraction recovery rate, matrix effect, and stability based on China Food and Drug Administration: Guideline on Bioanalytical Method Validation, Chinese Pharmacopoeia (2015)^[18].

Specificity

Six different drug-free RBC lysate and QC samples containing analytes were used to compare after sample pre-treatment. According to the obtained chromatographic peaks, no significant response was observed for MTX and MTX metabolites at the retention time.

Lower Limit Of Quantitation (Lloq)

Adding different concentrations of MTXPGn into the blank RBC lysate, the LLOQ is defined as the lowest concentration with CV < 20% and signal-to-noise ratio (S/N) > 10.

Linearity

To assess the linearity, standard curves in five consecutive runs were constructed by plotting the nominal concentrations against MTXPGn to internal standard area ratios with a weighted least square linear regression model. The final concentration of standard curve ranged 1 from 500 nM. The square correlation coefficient (R²) of each calibration curve should be greater than 0.99, so the linearity is considered acceptable.

The Precision And Accuracy

The precision and accuracy within and between batches were measured with blank RBC lysate at four different concentration levels, such as quantitative lower limit, low, medium and high quality control concentration, and the precision and accuracy within and between batches of three batches were evaluated within at least three days. The precision was determined by calculating the coefficient of variation (CV%) of repeated measurements. The accuracy is determined by the difference between the measured concentration and the spiked concentration, which is expressed as error.

Extraction Recovery

Before and after sample preparation, MTXPGn were added to different batches of mixed blank RBC lysate to determine the recovery rate of sample preparation. The recovery rate is calculated as follows: the recovery rate (%) of analyte spiked into RBC before sample preparation = peak area of analytes/peak area of spiked analytes in supernatant obtained from RBC after sample preparation) ×100%.

Matrix Effects (Dup: Abstract ?)

The matrix effect was measured at the following two sets of QC samples. Set 1: peak areas in postextraction spiked samples. Set 2: peak areas in samples spiked with the same amounts of analytes at four QC levels. The matrix effect of the method was calculated as Set 1/ Set 2*100%.

Investigation Of Stability

The stability of analytes were evaluated by taking four types of QC samples under various conditions: at 4℃ for 24 hours, at room temperature for 16 hours, freeze-thaw cycle at -80℃ for 3 times, and at -80℃ for 30 days. The deviation of QC samples should be less than 15%.

Drug Monitoring

Samples of 89 pediatric ALL patients were obtained from the study of South China Children's Leukemia Group (SCCLG-ALL-2016 Protocol) ^[19]. These children were in maintenance treatment and received similar doses of treatment (median, 15.03 mg/m² /week; 1.25-35 mg/ week), with a median dose of 279 days; Range, 105–864 days). The study was approved by the children or their families and the local ethics committee (Ethical numbering: 2022-KY-011).

Statistical Data

Xcalibur 2.0 software was used to analyze MTX concentration and MTXPG2-7 concentration in erythrocyte lysate. Then, SPSS 19.0 statistical software package was used for statistical test. The concentration levels of patients with different genotypes were compared according to nonparametric test, and p < 0.05 was the threshold of significant difference in data. GraphPad Prism 8.0 was used for drawing.

Speciality and linearity

The calibration curves of analytes were yielded with nine increasing concentration levels and showed good linearity over the range of 1-500 nM (Table 2 for linear regression equations).. The lower quantitative limit of MTXPGn were 1 nM. No interference was detected at the corresponding peak time in the blank samples.

Table 2

Typical calibration equations of methotrexate and its active metabolites.
Compound	Linear equations (1/X, R²)
MTX	y = 0.00605x + 0.0308, 0.9985
MTX-PG2	y = 0.0192x + 0.00310, 0.9918
MTX-PG3	y = 0.0123x + 0.00253, 0.9949
MTX-PG4	y = 0.0139x + 0.00666, 0.9950
MTX-PG5	y = 0.0125x-0.0291, 0.9909
MTX-PG6	y = 0.000373x-0.000341, 0.9950
MTX-PG7	y = 0.000122x-0.000378, 0.9978

The Precision, Accuracy And Lloq

The precision and accuracy of this method were evaluated at four different QC concentrations (3, 200 and 400 nM) and LLOQ concentration (1 nM). It is expressed by the percent variation coefficient of precision (%RSD). Intra-day and inter-day precision were less than 15%. Specific data were shown in Table 3. A typical LLOQ chromatogram was shown in Fig. 2.

Recovery

The extraction recovery of four different concentrations of MTX and MTXPG_2 − 7 were determined, as shown in Table 3. The average recoveries of MTXPG_2 − 7 were 81.09%, 74.75%, 69.29%, 64.92%, 71.44% and 62.51% respectively.

Matrix Effects

Matrix effects of MTX and MTXPG2-7 at different QCs were shown in Table 3, among of which all CV% were less than 15%.

Table 3

Precision/accuracy, recovery and matrix effect of erythrocyte methotrexate and polyglutamylated metabolites.
Compound	QCs (nmol/L)	Intra-batch		Inter-batch		Recovery (Mean ± SD, CV%)	Matrix effect (Mean ± SD, CV%)
Compound	QCs (nmol/L)	CV (%)	RE (%)	CV (%)	RE (%)	Recovery (Mean ± SD, CV%)	Matrix effect (Mean ± SD, CV%)
MTX	LLOQ	2.44	10.54	4.16	5.47	77.83 ± 9.31,11.96%	73.56 ± 7.70,10.47%
	LQC	5.73	4.91	1.73	2.86	84.84 ± 5.00,5.90%	81.37 ± 5.76,7.08%
	MQC	2.50	7.09	0.54	6.43	99.75 ± 2.89,2.90%	72.43 ± 4.95,6.85%
	HQC	9.05	2.94	0.41	2.46	95.71 ± 5.96,6.23%	81.55 ± 7.22,8.85%
MTXPG₂	LLOQ	9.72	0.56	3.67	3.24	76.73 ± 9.23,12.03%	79.29 ± 9.94,12.54%
	LQC	9.06	-9.94	6.99	-5.26	97.96 ± 4.91,5.01%	89.90 ± 13.10,14.57%
	MQC	9.85	-2.01	2.02	-0.59	81.09 ± 3.92,4.83%	96.33 ± 1.90,1.98%
	HQC	3.71	9.04	4.90	5.38	65.89 ± 7.31,11.09%	87.30 ± 8.44,8.67%
MTXPG₃	LLOQ	8.62	3.65	2.26	5.85	77.13 ± 2.59,3.35%	72.18 ± 5.60,7.76%
	LQC	5.47	6.40	5.05	1.80	69.05 ± 4.63,6.71%	73.68 ± 2.19,2.99%
	MQC	5.87	-2.78	6.11	-0.49	74.75 ± 1.30,1.75%	74.07 ± 5.76,7.77%
	HQC	8.02	5.84	1.75	3.74	67.10 ± 5.99,8.91%	75.77 ± 3.73,4.92%
MTXPG₄	LLOQ	6.05	3.47	2.13	3.69	72.11 ± 3.66,5.07%	76.27 ± 3.33,4.37%
	LQC	2.04	0.85	2.37	0.18	69.02 ± 4.98,7.21%	72.67 ± 4.43,6.09%
	MQC	7.59	7.76	6.90	0.87	69.29 ± 3.35,4.82%	71.37 ± 5.27,7.39%
	HQC	4.24	1.72	3.01	2.82	64.46 ± 6.64,10.30%	74.09 ± 5.64,7.61%
MTXPG₅	LLOQ	5.80	1.08	2.36	-1.29	66.39 ± 9.44,14.21%	71.29 ± 6.29,8.82%
	LQC	10.47	3.65	5.45	0.93	76.57 ± 9.62,12.56%	70.84 ± 7.61,10.74%
	MQC	5.72	4.86	5.81	4.05	64.92 ± 7.76,11.94%	63.24 ± 3.74,5.92%
	HQC	7.10	4.76	1.43	6.40	64.89 ± 6.01,9.27%	71.44 ± 5.72,8.01%
MTXPG₆	LLOQ	2.48	0.45	2.87	-0.93	64.85 ± 2.66,4.41%	71.93 ± 10.63,14.77%
	LQC	3.77	-2.56	2.60	0.35	68.32 ± 4.62,6.76%	69.08 ± 7.77,11.24%
	MQC	10.07	8.76	6.44	2.24	71.44 ± 5.88,8.22%	68.76 ± 6.74,9.80%
	HQC	5.89	1.68	0.64	1.73	72.32 ± 5.62,7.76%	73.20 ± 10.71,14.63%
MTXPG₇	LLOQ	8.13	7.88	5.05	1.95	72.37 ± 10.60,14.65%	69.57 ± 6.49,9.33%
	LQC	11.14	-7.71	6.64	-4.83	66.46 ± 7.60,11.43%	71.43 ± 10.07,14.11%
	MQC	9.29	6.12	7.62	1.53	62.51 ± 9.31,14.89%	65.66 ± 3.50,5.33%
	HQC	4.68	10.56	6.33	3.19	64.69 ± 8.59,13.29%	68.05 ± 6.67,9.79%

Stability

The results showed that MTX and MTXPG_2 − 7 were stable under the above conditions, with RSD < 15% (Table 4).

Table 4

Stability of analytes under different conditions (Mean ± SD, CV%).
Compound	QCs	Stability (%)
Compound	QCs	4°C (24 h)	Room temperature (16 h)	Three freeze-thaw cycles	−80°C (30 days)
MTX	LLOQ	2.15 ± 0.10, 4.61%	2.07 ± 0.18, 8.63%	2.16 ± 0.05, 2.47%	2.16 ± 0.08, 3.92%
MTX	HQC	219.12 ± 8.79, 4.01%	219.82 ± 4.66, 2.12%	206.19 ± 18.98, 9.21%	222.94 ± 7.24, 3.25%
MTXPG₂	LLOQ	2.12 ± 0.06, 2.83%	2.11 ± 0.19, 9.16%	2.17 ± 0.05, 2.51%	2.12 ± 0.03, 1.64%
	HQC	217.63 ± 569, 2.61%	214.42 ± 15.57, 5.42%	214.57 ± 5.43, 2.53%	215.64 ± 11.18, 5.19%
MTXPG₃	LLOQ	2.25 ± 0.04, 1.83%	2.05 ± 0.21, 10.41%	2.02 ± 0.01, 0.36%	2.22 ± 0.13, 5.76%
	HQC	223.37 ± 6.26, 2.80%	215.27 ± 16.51, 7.67%	209.41 ± 11.25, 5.37%	212.92 ± 13.22, 6.21%
MTXPG₄	LLOQ	2.25 ± 0.04, 1.83%	2.29 ± 0.15, 6.36%	2.07 ± 0.04, 2.01%	2.33 ± 0.06, 2.45%
	HQC	223.37 ± 6.26, 2.80%	203.09 ± 0.52, 0.25%	209.07 ± 7.88, 3.77%	206.20 ± 2.67, 1.30%
MTXPG₅	LLOQ	2.07 ± 0.06, 1.26%	2.24 ± 0.12, 5.29%	2.03 ± 0.01, 0.45%	2.17 ± 0.17, 7.86%
	HQC	229.27 ± 2.90, 1.26%	202.56 ± 0.42, 0.21%	209.34 ± 9.84, 4.70%	203.84 ± 1.24, 0.61%
MTXPG₆	LLOQ	2.21 ± 0.09, 3.97%	1.98 ± 0.17, 8.56%	2.25 ± 0.04, 1.78%	2.16 ± 0.24, 11.07%
	HQC	201.41 ± 18.16, 9.01%	222.25 ± 5.54, 2.49%	222.08 ± 4.28, 1.93%	218.64 ± 1.91, 0.87%
MTXPG₇	LLOQ	2.16 ± 0.11, 5.05%	2.06 ± 0.20, 9.81%	2.21 ± 0.09, 4.16%	2.07 ± 0.29, 14.22%
	HQC	200.95 ± 0.34, 0.17%	209.86 ± 19.11, 9.11%	215.31 ± 22.06, 10.25%	217.51 ± 19.11, 8.79%

Application Of Lc-ms/ms Method In Pediatric All Patients

The total average concentration of 89 patient samples obtained from SCCLG-ALL-2016 protocol study was 139.0 ± 54.3 nM, and the average values of MTX, MTXPG₂, MTXPG₃, MTXPG₄, MTXPG₅, MTXPG₆ and MTXPG₇ were (35.3 ± 28.1) nM, (21.3 ± 9.2) nM, (21.3 ± 9.2) nM, (21.3 ± 9.2) nM, (21.3 ± 9.2) nM, (21.3 ± 9.2) nM, (21.3 ± 9.2) nM (Fig. 3). The main glutamic acid form of MTX in RBC is MTXPG₃, which accounts for 37% of the total MTX content in RBC. The polymorphism of MTHFR C677T was detected in 89 cases, including 74 cases of CC type, 10 cases of CT type and 5 cases of TT type. To further verify the relationship between the gene polymorphism and MTXPGn concentration, the concentrations of MTX, MTXPGn and MTXPGsum of different genotypes were compared, and found that there was no obvious difference in the measured concentrations among different genotypes (Fig. 4).

In the present study, a sensitive bioassay to simultaneous determination of erythrocyte methotrexate and polyglutamylated metabolites was established and applied to monitor the MTXPGn profiles during maintenance therapy pediatric ALL patients. The dose of MTX is not the main factor causing the cytotoxicity, but the MTXPG. MTX enters cells and forms MTXPG under the action of enzymes. The aggregation amount and chain length of MTXPG are the main factors causing the cytotoxicity. Studies have shown that after MTX enters effector cells, glutamate residues are connected in turn under the action of folylpolyglutamate synthetase (FPGS) to form MTXPGs. Because only the MTX prototype can be excreted by ATP-binding cassette (ABC) protein family, but the MTXPGs that cannot be transformed into MTX prototype cannot be excreted, MTXPG_2 − 7 stays in the cells for a longer time. In the cell, MTXPG_2 − 7 not only inhibits dihydrofolate reductase (DHFR), but also has higher affinity for key enzymes of folate metabolism (such as thymidylate synthase (TS) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) formyltransferase) than MTX prototype ^[20]. Therefore, the efficacy and adverse reactions of MTX mainly depend on the concentration levels of MTXPG_2 − 7 in effector cells.

There are many methods to determine the blood concentration of MTX. The commonly used methods include chemiluminescence immunoassay (CLIA), fluorescence polarization immunoassay and HPLC. Although immunoassay method is relatively simple and highly automated, it costs more, susceptible to the interference of metabolites and produces cross reaction, so the accuracy of the determination results is not meet the satisfaction in clinic^{[14, 21]}. HPLC ^[22–23] and LC-MS/MS have strong specificity to differentiate target drugs and metabolites, while LC-MS/MS can work at a higher sensitivity than HPLC. In this study, in order to establish a stable and accurate LC-MS/MS method to detect the concentrations of MTX and MTXPG_2 − 7 in RBCs, the mass spectrometry and liquid phase conditions were optimized. The precursor/product ions of MTXPGn and IS were optimized by using syringe pump infusion directly in the mass spectrometer of each standard stock solutions. A comparison of signal ESI ionization intensity under negative and positive ion mode showed that, the [M + H]⁺ ion and main fragment ions in positive ion mode were stable and with a higher intensity than that in negative mode. Selective Reaction Monitoring(SRM) scanning mode is selected for biological sample analysis, which can monitor the target fragment ions as much as possible. Therefore, the optimized mass spectrometry conditions were set as follows: spray voltage: 3500 V, evaporation temperature: 350℃, sheath air pressure: 30 psi, auxiliary air pressure: 10 psi, temperature of ion transfer tube: 350℃.

Considering that the parent structure of the compounds to be tested is consistent, ion saturation inhibition may occur during ion fragmentation. Therefore, the compounds to be tested were separated at different times as far as possible to peak during the optimization of the liquid phase method. Different mobile phase system of methanol, acetonitrile, water and various kinds of additives (ammonium acetate buffer, formic acid, etc.) were used to optimize the liquid phase conditions. Typically, it was obvious that after adjusting the pH of water mobile phase, the chromatographic behaviors showed a better retention for each analyte, shorten running time and reduced peak tailing. A stable separation was achieved for structural analogs after eluting in a different order. Therefore, the liquid phase conditions are optimized as follows: (A) 2 mM ammonium acetate, and the pH is adjusted to 10 with 25% ammonia water; (B) Methanol. The chromatographic separation allows fast analysis with a total running time of 13.0 min for seven analytes, making them good candidates for clinical application. The linear range of this method was 1-500 nM, the intra-day precision variation coefficient was 2–11%, and the inter-day precision was 0.4-8%. The recovery rate ranged from 62.51–99.75%, and the relative matrix effect varied from 63.24–96.33%.

In this study, the concentrations of MTX and its metabolites in samples from 89 pediatric ALL patients were detected. ALL the patients were in the maintenance treatment period, and they were treated with MTX steadily. The results showed that the total average concentration was close to that reported^[24], and reached the target concentration during maintenance treatment, which confirmed that this research method was reliable. Among the metabolites, the content of MTXPG₃ is the highest, which confirms that MTX is the main glutamic acid form in RBCs. Methylenetetrahydrofolate reductase (MTHFR) is one of the key enzymes involved in catalyzing methylenetetrahydrofolate to produce methyltetrahydrofolatebin folate metabolism pathway, and it is also one of the key enzymes for MTX to exert pharmacological effects. Its gene polymorphism may be related to the blood drug concentration and adverse drug reactions of MTX after chemotherapy ^[25]. Therefore, this study also detected the concentrations of MTX and its metabolites under three different genotypes of this gene, but it was not found that this gene polymorphism affected the concentration of MTX in RBC.

To sum up, this study established a LC-MS/ MS method for detecting MTX in RBCs, and optimized its mass spectrum and liquid phase conditions to shorten the sample pretreatment and analysis time, which has good clinical practicability. In addition, the method has been successfully applied in 89 clinical patients, and the concentration and distribution of MTXPGn in these samples are basically consistent with those reported in the literature, which further confirms its reliability and accuracy.

MTX

Methotrexate

Acute lymphoblastic leukemia

MTXPG

Methotrexate polyglutamates

UHPLC-MS/MS

Ultra high performance liquid chromatography-tandem mass spectrometry

Rheumatoid arthritis

TDM

Therapeutic drug monitoring

HPLC

High performance liquid chromatography

LC-MS/MS

Liquid chromatography-tandem mass spectrometry

Aminopterin

HClO₄

Perchloric acid

ESI

Electrospray ionization

Quality control

LLOQ

Lower limit of quantification

FPGS

folylpolyglutamate synthetase

ABC

ATP-binding cassette

DHFR

Dihydrofolate reductase

Thymidylate synthase

AICAR

5-aminoimidazole-4-carboxamide ribonucleoside

CLIA

Chemiluminescence immunoassay

SRM

Selective Reaction Monitoring.

Acknowledgements

Not applicable.

Authors’ contributions

JW participated in the design of the study, involved in the sample preparation, carried out the solid phase extraction and drafted the manuscript. YG involved in the sample preparation, participated in the gradient chromatographic separation and drafted the manuscript. MQ performed the statistical analysis and drafted the manuscript. SY and CG participated in the gradient chromatographic separation. SL and MH participated in the sample preparation. YZ and JF participated in the clinical data collection. XW and DZ conceived of the study, participated in its design and coordination. All authors read and approved the final manuscript. All contributors who do not meet the criteria for authorship should be listed in an acknowledgements section.

Funding

This study was sponsored by the National Nature Science Fund of China, Grant No. 82020108031, 82104290, 81730103, 81700495, and 81870382; the National Key Research and Development Program, Grant No. 2016YFC0905003; and the 111 Project, Grant No. B16047.

Availability of data and materials

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

Ethics approval and consent to participate

The studies involving human participants were reviewed and approved by the Ethics Committee of Sun Yat-sen Memorial Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

None of the authors have competing interests.

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No competing interests reported.

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An optimized UHPLC-MS/MS method for simultaneous determination of erythrocyte methotrexate and polyglutamylated metabolites: Application in pediatric patients with acute lymphoblastic leukemia

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Abstract

Figures

Introduction

Methods

Materials

Lc-ms/ms Conditions

Preparation Of Stock Solution And Working Solution

Preparation Of Erythrocyte Lysate

Preparation Of Standard And Quality Control Samples

Sample Pretreatment

Methodological Verification

Specificity

Lower Limit Of Quantitation (Lloq)

Linearity

The Precision And Accuracy

Extraction Recovery

Matrix Effects (Dup: Abstract ?)

Investigation Of Stability

Drug Monitoring

Statistical Data

Results

Speciality and linearity

The Precision, Accuracy And Lloq

Recovery

Matrix Effects

Stability

Application Of Lc-ms/ms Method In Pediatric All Patients

Discussion

Abbreviations

Declarations

References

Additional Declarations

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