A reversed-phase high performance liquid chromatography method for quantification of methotrexate in cancer patients serum

Journal of Chromatography B, 1002 (2015) 107–112

Contents lists available at ScienceDirect

Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

A reversed-phase high performance liquid chromatography method for quantification of methotrexate in cancer patients serum Yuan-dong Li ∗ , Yan Li, Ning-sheng Liang, Fan Yang, Zhi-peng Kuang Research Department, Affiliated Tumor Hospital of Guangxi Medical University, Nanning, 530021 Guangxi, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 15 April 2015 Received in revised form 11 August 2015 Accepted 12 August 2015 Available online 20 August 2015 Keywords: Method validation HPLC Methotrexate Silver nitrate Serum

a b s t r a c t A simple, rapid and sensitive reversed-phase high performance liquid chromatography (HPLC) method has been developed for the determination of methotrexate in human serum. After deproteinization of the serum with 40% silver nitrate solution, methotrexate and internal standard (IS) were separated on a reversed-phase column with a mobile phase consisting of 10 mM sodium phosphate buffer (pH6.40)methanol (78:22%, v/v) and ultraviolet detection at 310 nm. The linearity is evaluated by a calibration curve in the concentration range of 0.05–10.0 ␮g/mL and presented a correlation coefficient of 0.9995. The absolute recoveries were 97.52 ± 3.9% and 96.87 ± 3.7% for methotrexate and ferulic acid (internal standard), respectively. The intra- and inter-day precision were less 6.19 and 5.89%, respectively (n = 6). The limit of quantitation was 0.02 ␮g/mL and the limit of detection was 0.006 ␮g/mL. The complete analysis was achieved less than 10 min with no interference from endogenous components or 22 examined drugs. This method was validated by using serum samples from high-dose methotrexate treated patients with osteosarcoma, breast cancer, acute leukemia and lymphoma. The method was demonstrated to be a simple, rapid and reliable approach in quantification of methotrexate in serum samples from patients with high-dose methotrexate therapy. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Methotrexate (MTX; Fig. 1) is an antifolate cytotoxic agent used to treat certain types of hematological cancers, solid tumors, and rheumatoid arthritis. High-dose methotrexate (HDMTX) has for decades been in clinical use as a cytotoxic drug for solid tumors and leukemias [1,2]. HDMTX has many serious toxic effects, such as myelosuppression, hepatic, renal and pulmonary disorders [3,4]. For safe and effective use of HDMTX, certain precautions should be followed. Routine monitoring of MTX concentrations in serum with early detection of abnormal clearance has permitted countermeasures, such as adjustments of leucovorin doses and intensified hydration regimes, to prevent excessive host toxicity [5,6]. Therefore, the routine monitoring of MTX concentrations in serum is important in guiding leucovorin rescue and is considered to be imperative for both patient safety and evaluation of therapeutic concentrations of MTX [5,7].

∗ Corresponding author at: Research Department, Affiliated Tumor Hospital of Guangxi Medical University, 71 Hedi Road, Nanning, 530021 Guangxi, People’s Republic of China. Fax: +86 771 5310593. E-mail address: [email protected] (Y.-d. Li). http://dx.doi.org/10.1016/j.jchromb.2015.08.017 1570-0232/© 2015 Elsevier B.V. All rights reserved.

Several chromatographic methods for determination of MTX in serum have been reported [8–17]. Among these methods, high sensitivity tandem mass spectrometry had been employed for MTX monitoring [8]. However, tandem mass spectrometry facilities are not always available as standard equipment in hospital laboratories, thereby limiting its application in clinical routines. Consequently, several HPLC methods have been developed to determine the levels of MTX in human serum following clean-up procedures, such as solid-phase extraction or protein precipitation [9–17]. Among these methods, solid-phase extraction method is the most used method, but it requires tedious solid phaseextraction procedures. Protein precipitation method usually uses trichloroacetic acid and shows lower recovery rate than solid-phase extraction methods [17]. There is still a need for a sensitive, rapid and inexpensive method broadly applicable to clinical routines for therapeutic drug monitoring of MTX. The aim of this study was to develop a simple, rapid and highly specific and sensitive HPLC method for quantitation MTX in serum samples after deproteinization and clean-up steps. The method was validated in a cohort of routine drug monitoring in different cancer patients under HDMTX treatment. This method involved the isolation of MTX from 200 ␮L serum by deproteinization by Silver nitrate and subsequent HPLC-ultraviolet quantification of MTX. This study included the evaluation of several frequently co-

108

Y.-d. Li et al. / J. Chromatogr. B 1002 (2015) 107–112

was added to precipitate serum proteins. The tubes were vigorously mixed for 1 min then heated at 45 ◦ C for 5 min to optimize the precipitation. Subsequently, 25 ␮L of 50 % (w/v) potassium iodide solution was added to the mixture, then the mixture was vortexed again for 1 min and centrifuged at 15,000 × g for 10 min. The supernatant solution (20 ␮L) was injected into the chromatographic system for analysis. 2.5. Method validation Fig. 1. Molecular structures of methotrexate and ferulic acid.

administered drugs to minimize errors resulting from the possible interference of these drugs with MTX, and thus, to enhance valuable analytical properties such as specificity. 2. Experimental 2.1. Reagents and chemicals Methotrexate, ferulic acid (internal standard, IS; Fig. 1), hydrocortisone, gentamycin, caffeine, ibuprofen, acyclovir, etoposide, allopurinol, 5-flurouracil, aspirin, paracetamol, dexamethasone, prednisolone, caspofungin, amoxicillin, aztreonam, clarithromycin, folic acid, gentamycin, levofolinate, methylprednisolone, naproxen, omeprazole, salicylate, cytarabine, aminopterin, acyclovir, inosine, ferulaic acid, silver nitrate, cisplatin, potassium dihydrogen phosphate and potassium iodide were analytical-reagent (AR) grade (Sinopharm Chemical Regent Corporation, Beijing, China). HPLC-grade methanol was obtained from Merck (Darmstadt, Germany). Deionized water was obtained from a Milli-Q Ultra-Pure Water System (Millipore, Bedford, MA). 2.2. Chromatographic conditions The HPLC system (Shimadzu, Japan) consisted of a CBM-20 pump, a SIL-20 autosampler, a CTO-20A column oven and SPD20A UV/VIS detector set at 310 nm. Samples were analyzed on a Zorbax-ODS C18 column (4.6 × 250 mm, 5 ␮m, Shimadzu), protected by a guard column (4.6 × 15 mm i.d.) of the same material. The mobile phase (10 mM sodium phosphate buffer (pH6.40): methanol, 78:22% (v/v) was vacuum degassed and filtered through a 0.2 ␮m Millipore membrane filter before use. The flow-rate was 1.0 mL/min and chromatography was performed at 35 ◦ C temperature. The injection volume was 20 ␮L for all injections.

The method was validated for specificity, linearity, precision, accuracy, and recovery according to the Drug Evaluation and Research guideline recommended by the US Food and Drug Administration Center for bioanalytical method validation [18]. 2.5.1. Specificity Specificity was determined by testing 6 drug-free serum samples from seven different healthy volunteers for interference with the analyte and the IS. Furthermore, the possibility of chromatographic interference with the most frequently co-administered drugs was examined by analyzing several commercially available medications. The following substances were dissolved in the appropriate solvent for each medication filtered and analyzed: hydrocortisone, amoxicillin, aztreonam, caspofungin, clarithromycin, doxycycline, gentamycin, methylprednisolone, prednisolone, naproxen, omeprazole, etoposide, 5-flurouracil, caffeine, dexamethasone, folic acid, paracetamol, salicylate, levofolinate, acyclovir and allopurinol. 2.5.2. Recovery, limit of quantitation and limit of detection The extraction recoveries of MTX in human serum at three QC levels were determined by comparison of the peak area of the QC samples (n = 3) from extracted samples with standard solutions and without extraction procedure at equivalent concentrations. The method recovery was determined for QC samples spiked at the 3 concentrations by comparing the calculated concentrations with the corresponding spiked concentrations. The limit of quantitation (LOQ) of the method was defined as the point where the signal to noise ratio was equal to 10. The limit of detection (LOD) was defined as the point where the signal to noise ratio equaled 3.

2.3. Preparation of standard solutions, calibration standards and quality control samples

2.5.3. Linearity The calibration curve was generated with the concentrations ranging from 0.05 to 10.0 ␮g/mL. The calibration curve was constructed via linear regression, and the linear range of the analyte was calculated using the summed peak area ratio of MTX to that of the internal standard (ferulic acid).

Stock solution of MTX was prepared by dissolving 25.0 mg of MTX in 50 mL of in mobile phase. The IS solution was prepared by dissolving 20.0 mg of ferulic acid in 100 mL of the same solvent. Working solutions of each analyte at different concentration levels were freshly prepared by appropriate dilution with the previous solvent mixture in amber-glass vials. Aliquots of these working solutions together with 4 ␮L IS were added to blank human serum prior to prepare calibration standards resulting in 0.05, 0.1, 0.5, 1.0, 2.0, 5.0 and 10 ␮g/mL, respectively. Low, medium and high quality control (QC) samples were similarly prepared containing MTX concentrations in serum of 0.1, 1.0 and 5.0 ␮g/mL, respectively.

2.5.4. Precision and accuracy Intra-day precision and accuracy was performed with six replicates of the low (0.1 ␮g/mL), medium (1.0 ␮g/mL) and high (5.0 ␮g/mL) QC samples, whereas inter-day evaluation was assessed by the analysis of one replicate at each QC sample concentration on six different day. Intra- and inter-day precisions were determined by assessing the measurement of QC samples at low, medium and high concentration. Precisions were expressed by the coefficient of deviation (CV%) was defined as the ratio of the standard deviation to the mean, while accuracy (%) was expressed as the percentage of observed value to true value.

2.4. Sample preparation

2.6. Biological samples

Serum samples (200 ␮L) were placed in polypropylene centrifuge tubes followed by 5 ␮L of ferulic acid solution (200 ␮g/mL) as internal standard (IS). 20 ␮L of 40% (w/v) silver nitrate solution

Blood samples were obtained from patients diagnosed with osteosarcoma, breast cancer, acute leukemia and lymphoma (Department of Clinical Oncology, Affiliated Tumor Hospital of

Y.-d. Li et al. / J. Chromatogr. B 1002 (2015) 107–112

109

Table 1 Characteristics and features of patients enrolled in the present study. Cases

Disease

Age (year)

Sex (M/F)

Weight (kg)

BSA (m2 )

Dose (mg)

Patient A Patient B Patient C Patient D Patient E Patient F

Breast cancer Acute leukemia Lymphoma Lymphoma Osteosarcoma Osteosarcoma

50 7 45 40 34 35

M M F M F M

65 25 48 70 51 73

1.75 0.85 1.54 1.93 1.63 1.71

2625 2550 4620 5790 9780 10260

Infusion time (h) 6 16 20 20 8 8

Toxicity development (yes/no) No No No No No No

BSA: body surface area.

Guangxi Medical University) who were receiving HDMTX as part of a treatment protocol approved by the University Hospital Scientific Review Board between September 2013 and February 2015. All patients who participated in the study gave their written informed consent. During HDMTX treatment, patients received intensive hydration and urine alkalization, leucovorin rescue commenced 24 h following HDMTX treatment. Blood samples obtained at 0, 12, 24, 48 and 72 h when necessary, at the end of MTX infusion, were collected by venipuncture into 5 mL vacuum collection tubes. Serum was prepared by centrifugation at 3200 × g for 5 min; and were analyzed immediately. The patient characteristics are summarized in Table 1. 3. Results and discussion 3.1. Optimization of chromatographic conditions The formulation of the mobile phase is an important factor for separating MTX from IS and endogenous components. During analytical method development, we found that the retention time of MTX was significantly affected by pH value. When the pH value of sodium phosphate buffer was greater than 7.00, MTX peak could be delay. However, if the pH value was decreased to 5.00, endogenous components from serum would interfere with the MTX peak. Finally, the sodium phosphate buffer (pH 6.40) was adopted to prepare the mobile phase to obtain a proper retention time and optimized shape of the MTX and IS peak. The chromatographic conditions were optimized through several trials to achieve good resolution and symmetric sharp peaks for both analytes and IS, as well as a short run time. During the optimization of mobile phase methanol–water and acetonitrile–water were investigated. A better separation was obtained using methanol–water compared with acetonitrile–water. And 10 mM sodium phosphate buffer (pH 6.40) as the modifier was adopted to avoid tailing. Fig. 2 shows representative chromatograms of a blank human serum sample (Fig. 2A), a standard solution of MTX (5.0 ␮g/mL) and the IS (5.0 ␮g/mL) (Fig. 2B), a blank serum sample spiked with MTX (5.0 ␮g/mL) and IS (5.0 ␮g/mL) (Fig. 2C) and a patient’s serum sample collected 24 h following MTX administration, spiked with IS (5.0 ␮g/mL) (Fig. 2D). The approximate retention times for MTX and IS were 5.00 and 5.81 min, respectively. Both MTX and IS eluted as symmetrical peaks and no interfering peaks were observed. The chromatographic condition developed in this work was simple and reproducible. It could exclude the interferences from other endogenous in serum and produce good-resolution analytic peaks. This method also made it possible for determining the concentration of serum MTX by HPLC within 10 min.

trichloroacetic acid [16] as protein precipitants was also common methods for sample preparation. But these methods show lower recovery rate and high background noise. We studied a protein precipitation method usually using acetonitrile, methanol, perchloric acid, trichloroacetic acid and silver nitrate as precipitating reagents. The use of 40% (w/v) silver nitrate solution was found to be most effective solution for the removal of protein and interfering compounds and recovery of MTX from human serum (Fig. 2). The protein precipitating effect of silver nitrate is a known fact. The mechanism is that the silver ions bind to the thiol groups on the protein to form silver protein complex. This work proved that using silver nitrate as precipitating agent was satisfactory for the separation of protein from serum. Compared with other precipitating method like perchloric acid [8], acetone [12] and trichloroacetic acid [16], the method of the use of 40% (w/v) silver nitrate solution was a superior sample preparation. 3.3. Optimization of the extraction and clean-up procedures The excessive silver nitrate may affect the lifetime of the HPLC column and the long-term quality of analysis. We studied precipitant of silver nitrate method using sodium chloride and potassium iodide as chemical reagents. The use of 50% (w/v) potassium iodide was found to be most effective for the removal of silver nitrate and recovery of MTX. The recover of MTX while using sodium chloride as precipitant was only 71.40 ± 5.3%. During preliminary experiments, medium quality control (QC) samples were placed in polypropylene centrifuge tubes followed by IS. Silver nitrate solution was added to precipitate serum proteins. The tubes were vigorously mixed. Thereafter, the mixtures were centrifuged. 25 ␮L of 50% (w/v) potassium iodide solution was added to the mixture before it was revortexed and centrifuged. The supernatant solution (20 ␮L) was injected into the chromatograph. The extraction recovery was only 56.54 ± 7.3%. Therefore, in the latter treatment, part of the drug is obviously co-precipitated with proteins, leading to reduced recovery compared to the current method. We found that it was very important to remove as much of the endogenous serum constituents as possible to achieve a low background signal. Using acetone as precipitating agent has been proved to be effective in separating protein from plasma [12]. However, it does not work for removing endogenous interfering compounds. There was still a need for further purified the supernatant by solvent extraction with a mixture of butanol and diethyl ether. The method developed in this work was much more effective in removing protein and other interfering compounds. 3.4. Selection of the internal standard

3.2. Optimization of the deproteinization Several chromatographic methods have been developed using different protocols for MTX separation in biological samples [8–17]. Among these methods, solid-phase extraction method is tedious procedures. Using perchloric acid [8], acetone [12] and

Regarding the IS selection, drugs such as cytarabine, aminopterin, acyclovir, inosine, ferulic acid and metronidazole were tested according to structural similarity. Adding internal standard which structural similar to the target analyte can improve quantification accuracy for MTX measurement. In this study, we

110

Y.-d. Li et al. / J. Chromatogr. B 1002 (2015) 107–112 Table 2 MTX determination in serum samples: intra-and inter-day precision and accuracy (n = 6). Quality control Low (0.1 ␮g/mL)

Medium (1.0 ␮g/mL)

High (5.0 ␮g/mL)

Intra-day assay Mean (␮g/mL) SD CV (%) Accuracy (%)

0.097 0.006 6.19 −3.00

1.01 0.048 4.75 1.00

4.98 0.049 0.98 −0.40

Inter-day assay Mean (␮g/mL) SD CV (%) Accuracy (%)

0.102 0.003 2.94 2.00

0.985 0.058 5.89 −1.50

4.97 0.073 1.47 −0.58

found that the retention times for cytarabine, aminoptern, acyclovir, inosine, metronidazole and ferulic acid were 3.51, 3.96, 3.97, 4.24, 7.23 and 5.80 min, respectively. Finally, ferulic acid was selected as the internal standard, because of being an appropriate retention time and a good resolution from MTX under the chromatographic condition. After selection of the internal standard, HPLC separation conditions with high selectivity and specificity were developed. 3.5. Validation of the method 3.5.1. Specificity Possible chromatographic interferences with co-administered drugs were tested. Antibiotics, pain killers, corticoids, antiemetic and anticancer drugs likely to be given to patients undergoing MTX chemotherapies were chromatographed under our experimental conditions. None of the drugs tested interfered with MTX or the IS (Table 3). 3.5.2. Recovery, limit of quantitation and limit of detection The mean absolute recovery of MTX from human serum at three concentrations was 97.52 ± 3.9% (n = 3 for each concentration). The mean absolute recovery of the IS was 96.87 ± 3.7% (n = 6). The method recoveries of MTX for QC samples at three concentrations were 97.81 ± 3.5% at low QC, 98.75 ± 4.5% at medium QC and 101.21 ± 4.5% at high QC, respectively. The LOD, where the signal to noise ratio equaled 3, was 0.006 ␮g/mL The LOQ, where the signal to noise ratio equaled 10, was 0.02 ␮g/mL. 3.5.3. Linearity of calibration curve The calibration curve of MTX in human serum was linear over the concentration range 0.05–10 ␮g/mL. The regression equation was Y = 1.0001X + 0.0096 (r = 0.9995, n = 6), where Y and X are the peak-area ratio and the concentration (␮g/mL in the serum). The mean precision (RSD) for the slope of MTX calibration curves was 5.6% (n = 6).

Fig. 2. HPLC chromatograms: bland serum (A); standard solution of 5.0 ␮g/mL MTX and IS (B); blank serum spiked with 5.0 ␮g/mL MTX and IS(C); serum sample of a patient F 24 h after MTX dosing spiked with IS (D); and serum sample of a patient B 12 h after MTX dosing spiked with IS (E).

3.5.4. Precision and accuracy On validation phase the precision and accuracy were determined by analyzing QC samples for three concentration levels. Each level was analyzed six times on the same day for intra-day precision and accuracy, and on six consecutive days for inter-day precision and accuracy. The results of intra-day (n = 6) and inter-day (n = 6) method validation are presented in Table 2. Intra-day precision was 6.19, 4.75, and 0.98% respectively, for the three levels tested. Respective accuracies were −3.00, 1.00 and −0.40% (n = 6). Interday precision was 2.94, 5.89 and 1.47% respective, for the three levels tested. Respective accuracies were 2.00, −1.50 and −0.58% (n = 6). These data indicate that the HPLC method is reliable both

Y.-d. Li et al. / J. Chromatogr. B 1002 (2015) 107–112 Table 3 Drugs frequently co-administered with MTX and detectable at UV 310 nm, characterized by retention time distinguishable from of MTX and IS. Drugsa

Retention times (min)

Methotrexate IS Aspirin Etoposide 5-Fu Caffeine Dexamethasone Folic acid Paracetamol Salicylate Levofolinate

5.00 5.81 16.96 6.77 4.72 4.69 4.16 3.57 2.82 2.80 2.48

111

lower MTX level that may present a risk for toxicity, thus ensuring effective and safe therapy for patients under HDMTX. 4. Conclusion Silver nitrate solution is an effective solution for removal of protein and interfering compounds and recovery of MTX from human serum. We have established a simple, rapid, isocratic, reversedphase HPLC method for the determination of MTX in human serum. The results confirm that this method can be applied for routine use in clinical investigation. Acknowledgements

a

The following drugs were also examined, though not detected, at UV 310 nm: hydrocortisone, ibuprofen, amoxicillin, aztreonam, caspofungin, clarithromycin, gentamycin, methylprednisolone, prednisolone, naproxen, omeprazole, aciclovir and allopurinol.

Table 4 Concentration changes of MTX in cancer patients serum with high-dose MTX therapy. Time (h)

0 12 24 48 72

Concentration (␮g/mL) Patient A

Patient B

Patient C

Patient D

Patient E

Patient F

65.26 1.542 0.105 0.014

25.39 1.213 0.101 0.010

34.77 1.274 0.255 0.023

36.82 1.465 0.323 0.021

236.54 15.70 1.727 0.442 0.012

199.21 8.928 0.874 0.184 0.008

within the same day and on different days. The intra- and inter-day precision and accuracy of the assay was satisfactory. The HPLC method developed in the present was a simple, rapid, specific, sensitive, precise, accurate, cost-effective and exempts the strict requirements for fluorescence spectrometry, tandem mass spectrometry, solid-phase and special fluorescent derivatization reagents [8–16]. Possible chromatographic interference between MTX and serum endogenous compounds and co-administered drugs has been precluded by sample pretreatment and chromatographic separation. So this method can be used in monitoring of HDMTX, especially in resource-limited settings. 3.6. Biological validation The clinical applicability of the method was examined by determining the concentration of MTX in human serum samples, obtained from cancer patients (osteosarcoma, breast cancer, acute leukemia and lymphoma) who had received HDMTX therapy. Table 4 shows the changes in the serum levels of MTX from 0, 12, 24, 48 and 72 h after the end of infusion with high-dose MTX therapy. The method described here has been successfully used to monitor the concentration of MTX in human serum after a therapeutic dose. Chromatograms of serum samples from a patient receiving 10,260 mg MTX, 24 h after the end of infusion, are shown in Fig. 2. HDMTX can result in severe toxicity if used inappropriately, so certain precautions should be applied to promote safe and effective use of HDMTX [16,17,19]. Monitoring of serum MTX level is very important to improve the safety of HDMTX therapy. Several reported studies have suggested that the cutoff points at which the drugs is considered adequately cleared and that are associated with the end of leucovorin rescue may range from 0.90 to 1.00 ␮mol/L (0.41–0.45 ␮g/mL) at 42–48 h [6,20] to 0.20 ␮mol/L (0.09 ␮g/mL) [2] and as low as 0.10 ␮mol/L (0.045 ␮g/mL) at 72 h post-MTX [2,19,21]. The present method accurately determines MTX concentrations as low as 0.044 ␮mol/L (0.02 ␮g/mL), which is below the

The work is supported by the Health Department of Guangxi Zhuang Autonomous Region (Grant No. 2012090). The authors would also like to express their gratitude to Miss Yan Feng for her technical support. References [1] C. Dupuis, C. Mercier, C. Yang, S. Monjanel-Mouterde, J. Ciccolini, R. Fanciullino, B. Pourroy, J.L. Deville, F. Duffaud, D. Bagarry-Liegey, A. Durand, A. lliadis, R. Favre, High-dose methotrexate in adults with osteosarcoma: a population pharmacokinetics study and validation of a new limited sampling strategy, Anti-Cancer Drugs 19 (2008) 267–273. [2] S. Zelcer, M. Kellick, L.H. Wexler, R. Gorlick, P.A. Meyers, The Memorial Sloan Kettering Cancer Center experience with outpatient administration of high dose methotrexate with leucovorin rescue, Pediatr. Blood Cancer 50 (2008) 1176–1180. [3] W.A. Bleyer, Methotrexate: clinical pharmacology, current status and therapeutic guidelines, Cancer Treat. Rev. 4 (1977) 87–101. [4] O.M. Al-Quteimat, M.A. Al-Badaineh, Practical issues with high dose methotrexate therapy, Saudi Pharm. J. 22 (2014) 385–387. [5] W.H. Isacoff, C.M. Townsend, F.R. Eiber, T. Forster, D.L. Morton, J.B. Block, High dose methotrexate therapy of solid tumors: observations relating to clinical toxicity, Med. Pediatr. Oncol. 2 (1976) 319–325. [6] R.G. Stoller, K.R. Hande, S.A. Jacobs, S.A. Rosenberg, B.A. Chabner, Use of serum pharmacokinetics to predict and prevent methotrexate toxicity, N. Engl. J. Med. 297 (1977) 630–634. [7] R.G. Stoller, K.R. Hande, S.A. Jacobs, S.A. Rosenberg, B.A. Chabner, Use of serum pharmacokinetics to predict and prevent methotrexate toxicity, New Engl. J. Med. 297 (1977) 630–634. [8] E. den Boer, S.G. Heil, B.D. van Zelst, R.J. Meesters, B.C. Koch, M.L. Te Winkel, M.M. van den Heuvel-Eibrink, T.M. Luider, R. de Jonqe, A U-HPLC-ESI–MS/MS-based stable isptope dilution method for the detection and quantitation of methotrexate in plasma, Ther. Drug Monit. 34 (2012) 432–439. [9] E. Begas, C. Papandreou, A. sakalof, D. Daliani, G. Papatsibas, E. Asprodini, Simple and reliable HPLC method for the monitoring of methotrexate in osteosarcoma patients, J. Chromatogr. Sci. 52 (2014) 590–595. [10] F. Albertioni, B. Pettersson, O. Beck, C. Rask, P.P. Seideman, C. Peterson, Simultaneous quantitation of methotrexate and its two main metabolites in biological fluids by a novel solid-phase extraction procedure using high-performance liquid chromatography, J. Chromatogr. B: Biomed. Appl. 665 (1995) 163–170. [11] E.A. McCrudden, S.E. Tett, Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly(styrene-divinylbenzene)column, J. Chromatogr. B: Biomed. Sci. Appl. 721 (1999) 87–92. [12] S. Emara, S. Razee, A. Khedr, T. Masujima, On-line precolumn derivatization for HPLC determination of methotrexate using a column packed oxidant, Biomed. Chromatogr. 11 (1997) 42–46. [13] S. Emara, H. Askal, T. Masujima, Rapid determination of methotrexate in plasma by high performance liquid chromatography with on-line solid-phase extraction and automated precolumn derivatization, Biomed. Chromatogr. 12 (1998) 338–342. [14] E. Begas, C. Papandreou, A. Tsakalof, D. Daliani, G. Papatsibas, E. Asprodini, Simple and reliable HPLC method for the monitoring of methotrexate in ostepsarcoma patients, J. Chromatogr. Sci. 52 (2014) 590–595. [15] W. Sadee, Antineoplastic agents: high-dose methotrexate and citrovorum factor rescue, Ther. Drug Monit. 2 (1980) 177–185. [16] M. Uchiyama, T. Matsumoto, T. Matsumoto, S. Jimi, Y. Takamatsu, K. Tamura, S. Hara, Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction, Biomed. Chromatogr. 26 (2012) 76–80. [17] L. Holmboe, A.M. Andersen, L. Morkrid, L. Slordal, K.S. Hail, High dose methotrexate chemotherapy: pharmacokinetics, folate and toxicity in osteosarcoma patients, Br. J. Clin. Pharmacol. 73 (2012) 106–114.

112

Y.-d. Li et al. / J. Chromatogr. B 1002 (2015) 107–112

[18] FDA Guidance for Industry Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), 2001. [19] B.C. Widemann, P.C. Adamson, Understanding and managing methotrexate nephrotoxicity, Oncologist 11 (2006) 694–703.

[20] M.V. Relling, D. Fairclough, D. Ayears, W.R. Crom, J.H. Rodman, C.H. Pui, W.E. Evans, Patient characteristics associated with high-risk methotrexate concentrations and toxicity, J. Clin. Oncol. 12 (1994) 1667–1672. [21] S.P. Treon, B.A. Chabner, Concepts in use of high-dose methotrexate therapy, Clin. Chem. 42 (1996) 1322–1329.