It was estimated that there were 385,700 new cases and 199,700 deaths of non-Hodgkin lymphoma (NHL) occurred in 2012 worldwide [
High-dose MTX (1-5 g/m2) could penetrate the blood-brain barrier, blood-eye barrier blood-testis barrier, and even the tumor cells to overcome the drug-resistance [
The standards including MTX, 7-OH-MTX, and MTX-d3 were purchased from Dalian Meilun Biotech Co., Ltd. (Dalian City, China. Purity >98% for MTX, 7-OH-MTX and MTX-d3. Lot No. MB1156-S, J0211A, J1213A). Chemical structures of MTX, 7-OH-MTX, and MTX-d3 are shown in Figure
The chemical structures of MTX, 7-OH-MTX, and MTX-d3.
All experiments were performed on an Agilent 6460 triple-quadrupole mass spectrometer equipped with an electrospray ionization source (Agilent Technology, MA, USA). The chromatographic separation was carried out on Agilent 1290 series ultra-high performance liquid chromatography, which contains a quaternary pump, an online degasser, an oven, and an autosampler. The instruments were operated with MassHunter software (Version 6.0.0, Agilent Technology, MA, USA).
Chromatographic separation was achieved on an Agilent ZORBAX C18 column (2.1 × 100 mm, 3.5
The product ion plots and fragmentor structures of MTX, 7-OH-MTX, and IS in positive mode. (a) MTX; (b) 7-OH-MTX; (c) IS.
The stock solutions of MTX and IS were prepared in methanol (including 0.1% ammonium hydroxide) both at 1.0 mg/mL, and for 7-OH-MTX, it was prepared in methanol-ammonium hydroxide-water (7:2:1, v/v/v) solution to obtain a final concentration of 1.0 mg/mL. All stock solutions were stored at −80°C until analysis. The work solutions of analytes and the IS (500 ng/mL) were freshly prepared by further diluting the stock solutions with methanol-water (1:9, v/v).
Calibrations standards were prepared by adding appropriate volume of the work solutions into blank human plasma at concentrations of 5, 10, 100, 200, 500, 2000, 5000, and 10000 ng/mL for MTX and 7-OH-MTX. The quality control (QC) samples were separately prepared in the same way to obtain concentrations of 10 (LQC, low), 500 (MQC, medium), and 5000 (HQC, high) ng/mL for MTX and 7-OH-MTX. All samples were stored at −80°C.
To a 100
Specificity was evaluated by comparing the chromatograms from six different lots blank samples with the spiked samples, and the corresponding responses less than 20% of LLOQ and 5% of IS were considered reasonable.
The recovery was assessed by comparing the peak areas of the spiked samples with the spiked postextraction samples at the same concentrations (n=3), and the matrix effect was estimated by comparing the peak areas of spiked postextraction samples to the water-substituted samples at the same concentrations (n=3). Both recovery and matrix effect were evaluated at low, medium, and high concentrations of the analytes. MTX and 7-OH-MTX were assessed at 10, 500, and 5000 ng/mL, and IS was investigated at a single concentration of 500 ng/mL.
The calibration curves of MTX and 7-OH-MTX ranged from 5 to 10000 ng/mL and nine calibration standards were prepared in this range for both analytes. The calibration curves were regressed by plotting the peak area ratio of analyte to IS versus corresponding concentrations using a 1/
The intraday and interday precision and accuracy were evaluated by analyzing five replicates of QC samples at three concentration levels in separated days (at least 2 days). Precision was expressed as the relative standard deviation (RSD%). The accuracy was determined as the deviation between the measured concentrations and the nominal concentrations. The RSD% for interday and intraday precision not more than 15% was rational. For intraday and interday accuracy, RE% (relative error) within ±15% was considered to be acceptable.
The stability, which included freeze-thaw stability, short-term stability, long-term, and bench-top stability of MTX, 7-OH-MTX, and IS in plasma, was investigated. Freeze-thaw stability was assessed after QC samples freeze-thaw for three cycles. Bench-top stability was assessed by analyzing the QC samples prepared and placed on the bench for 6 h. Long-term stability was tested by analyzing the QC samples stored at –80°C for 30 days. Short-term stability was evaluated by analyzing the extracted QC samples kept in the autosampler at 4°C for 24 h. The concentrations of analytes were calculated using accompanying constructed calibration curve.
Carry-over of MTX and 7-OH-MTX was also tested by injecting the highest calibration standard sample prior to injecting a blank sample, and the responses of analytes in blank samples not more than 20% of the LLOQ and 5% of the IS were considered to be acceptable.
The dilution effect was assessed by comparing the measured concentration to the nominal dilution concentration. Freshly prepared spiked samples with concentration beyond the highest calibration standard should be diluted by corresponding blank matrix into the linear range. Every dilution factor should be assessed at least for five times, and the RSD% and RE% should not more than 15% and within ±15%.
The experimental protocol was reviewed and approved by the Ethical Committee of Changzheng Hospital (Shanghai, China) and carried out in Changzheng hospital. Informed consent was signed by all the participants. Finally, 11 malignant lymphoma patients (male 5; female 6) in total were recruited and the samples were collected at 44, 68, and 92 h for every patient after administration of MTX using intravenous drip. The average age was 53.45 ± 9.75 (Mean ± SD) years. The dosage of MTX for the 11 patients was 0.77~3.50 g/m2 based on their clinical diagnosis. The samples were shaken slightly and then centrifuged at 3000 ×g for 10 min. The supernatant (plasma) was harvested and stored at −80°C until analysis.
LC-MS/MS method is based on the chromatography separation and mass spectrometry detection. An optimized chromatography separation and mass spectrometry detection could facilitate the drug quantification and analysis process. The first way to achieve the goal is to find a better mobile phase and the buffer substance, and the buffer substances could adjust the mobile phases pH and enhance the ionization efficiency to obtain a better peak symmetry and higher responses [
For LC-MS/MS development, the extraction and purification of analytes were most challenging and it was reported that approximately 61% of time was assigned to the extraction and purification process in a LC-MS/MS development [
Figure
The chromatograms of MTX, 7-OH-MTX, and IS under MRM mode: (a) blank sample; (b) blank plasma spiked with MTX (5 ng/mL); (c) blank plasma spiked with 7-OH-MTX (5 ng/mL); (d) blank plasma spiked with IS; (e) lower limit of quantification sample; (f) real sample collected at 44 h after the administration of MTX from one patient.
The recovery and matrix effect of MTX, 7-OH-MTX, and IS were presented in Table
Recovery and matrix effect for the analytes in human plasma (n=3).
Analyte | Nominal concentration (ng/mL) | Recovery | Matrix Effect | ||
---|---|---|---|---|---|
Mean ± SD | RSD | Mean ± SD | RSD | ||
(%) | (%) | (%) | (%) | ||
MTX | 10 | 92.76 ± 2.50 | 2.70 | 116.38 ± 1.33 | 1.15 |
500 | 97.87 ± 4.81 | 4.92 | 117.60 ± 7.07 | 6.01 | |
5000 | 92.47 ± 8.18 | 8.85 | 116.07 ± 1.26 | 1.08 | |
7-OH-MTX | 10 | 97.61 ± 4.69 | 4.80 | 102.96 ± 9.08 | 8.82 |
500 | 95.17 ± 3.51 | 3.69 | 97.90 ± 7.28 | 7.42 | |
5000 | 91.45 ± 4.27 | 4.67 | 99.57 ± 4.94 | 4.96 | |
IS | 500 | 100.50 ± 6.16 | 6.13 | 116.24 ± 3.06 | 2.63 |
Linearity and LLOQ are summarized in Table
Linearity regression parameters for MTX and 7-OH-MTX (n=5).
Analyte | Linearity Range | Calibration curve | | LLOQ | Measured Concentration | Accuracy |
---|---|---|---|---|---|---|
MTX | 5~10000 | | 0.9977 | 5 | 5.23 ± 0.21 | 4.57 |
7-OH-MTX | 5~10000 | | 0.9978 | 5 | 5.12 ± 0.56 | 2.35 |
The intraday and interday precision and accuracy of analytes at three concentration levels were summarized in Table
Intraday and interday precision and accuracy of MTX and 7-OH-MTX in human plasma (n=5).
Analyte | Nominal concentration | Intra-day(n = 5) | Inter-day(n = 15) | ||||
---|---|---|---|---|---|---|---|
Measured concentration | Precision | Accuracy | Measured concentration | Precision | Accuracy | ||
MTX | 10 | 9.48 ± 0.23 | 2.47 | -5.22 | 9.30 ± 0.32 | 3.41 | -7.05 |
500 | 514.84 ± 4.56 | 0.89 | 2.97 | 515.67 ± 9.49 | 1.84 | 3.13 | |
5000 | 5265.35 ± 92.88 | 1.76 | 5.31 | 5305.42 ± 118.95 | 2.24 | 6.11 | |
7-OH-MTX | 10 | 9.69 ± 0.66 | 6.86 | -3.15 | 9.70 ± 0.62 | 6.40 | -3.04 |
500 | 456.43 ± 8.69 | 1.90 | -8.71 | 469.16 ± 14.98 | 3.19 | -6.17 | |
5000 | 4772.84 ± 267.84 | 5.61 | -4.54 | 4772.93 ± 189.12 | 3.96 | -4.54 |
The analytes had acceptable stabilities after three freeze-thaw cycles, being in room temperature for 6 h, being stored at −80°C for 30 days, and being in autosampler at 4°C for 24 h. The results of all QC samples deviated within ±15%, as shown in Table
Stability of MTX and 7-OH-MTX in different conditions (n=5).
Analyte | Nominal concentration | three freeze-thaw cycles | Short-term | Long-term | Bench-top (RE/%) |
---|---|---|---|---|---|
MTX | 10 | -5.60 | -5.99 | -2.27 | -0.03 |
500 | 4.02 | 1.56 | -7.29 | 5.10 | |
5000 | 4.58 | 4.99 | 6.83 | 8.22 | |
7-OH-MTX | 10 | -0.91 | -5.96 | -9.10 | 7.22 |
500 | -8.04 | -8.66 | -12.91 | -0.77 | |
5000 | -7.02 | -5.80 | -5.26 | 2.41 |
The results displayed an obviously carry-over for MTX and 7-OH-MTX as the responses in the blank sample injected after the highest sample were more than 20% of the LLOQ responses. To solve this problem, a blank sample was added following a high concentration sample to elute the analytes residuals, and the carry-over of MTX and 7-OH-MTX was less than 20% of the LLOQ after the elution process.
For dilution effect assessment, a 20000 ng/mL spiked sample was freshly prepared and diluted for 5 times to 4000 ng/mL with blank plasma. After being assessed for 5 times, the RE% and RSD% of the dilution factor were less than 15% and within ±15%, which conformed to the criterions.
Finally, 49 samples were collected and quantitatively analyzed with this newly developed method. After administration, 22 samples were collected at 44 h; 21 sample were gathered at 68 h; and the other samples were harvested at 92 h. Research had suggested a safe concentration window for the MTX exposure [
Determined concentrations of MTX and 7-OH-MTX (Mean ± SD).
A sensitive, simple, and efficient UHPLC-MS/MS method has been successfully developed and validated for simultaneous determination of MTX and 7-OH-MTX in human plasma. The lower limit of quantification was 5 ng/mL for MTX and 7-OH-MTX. The analytical time was 3.5 min and the pretreatment procedure was developed based on a simple protein precipitation, which paved a way for high-throughput sample analysis. This method was applied on clinical samples from malignant lymphoma patients and the results were beneficial to avoid side effects after high-dose MTX therapy.
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest about the contents.
Xinxin Ren and Zhipeng Wang contributed equally to this work.
This work was supported by the Natural Science Foundation of Shanghai City, China (no. 17411972400); Shanghai Key Specialty Project of Clinical Pharmacy (2016-40044-002); and Important Weak Subject Construction Project of Shanghai Health Science Education (2016ZB0303).