A Validated HPLC-MS/MS Method for Quantification of Methotrexate and Application for Therapeutic Drug Monitoring in Children and Adults with Non-Hodgkin Lymphoma

Figures & data

Figure 1 The product ion plots of methotrexate and tinidazole (IS).

Figure 2 Typical MRM chromatograms of methotrexate and IS: (A) blank plasma sample; (B) blank plasma sample spiked with methotrexate at 5 ng/mL and IS at 100 ng/mL; and (C) real sample at 72 h after the start of MTX infusion.

Table 1 Evaluation of the Inter- and Intra-Batch Precision by the Proposed HPLC-MS/MS Method for Determination of MTX in Human Plasma (Mean±SD, n=5)

Preparation Concentration (ng/mL)	Intra-Day Precision			Inter-Day Precision
	Measured Concentration (ng/mL)	RSD (%)	Accuracy (%)	Measured Concentration (ng/mL)	RSD (%)	Accuracy (%)
8	7.79±0.48	6.10	97.35	8.04±0.56	6.99	100.46
80	77.60±5.95	7.67	97.00	81.11±5.01	6.22	101.46
800	801.80±33.09	4.13	100.23	817.04±39.73	4.86	102.15

Table 2 Extraction Recovery and Matrix Effect for Determination of MTX in Human Plasma Using the Proposed UPLC-MS/MS Method (Mean±SD, n=5)

Concentration Spiked (ng/mL)	Extraction Recovery		Matrix Effect
	(%)	RSD (%)	(%)	RSD (%)
8	93.98±4.50	4.78	103.25±5.24	5.07
80	82.20±8.30	10.10	102.69±4.92	4.79
800	90.19±5.16	5.72	105.28±10.80	10.26
IS	102.99±6.37	6.18	100.17±6.77	6.76

Table 3 Summary of Stability of MTX in Human Plasma (Mean±SD, n=5)

Condition Test	LQC (8 ng/mL)			MQC (80 ng/mL)			HQC (800 ng/mL)
	Mean Conc. (ng/mL)	RSD (%)	Accuracy (%)	Mean Conc. (ng/mL)	RSD (%)	Accuracy (%)	Mean Conc. (ng/mL)	RSD (%)	Accuracy (%)
Autosampler for 24 h	8.93±0.44	4.98	111.68	79.47±3.78	4.75	99.33	824.42±47.31	5.74	103.06
Room temperature for 6 h	7.45±0.20	2.64	93.06	76.84±3.52	4.58	95.98	731.00±33.52	4.59	91.38
4 °C for 24 h	7.92±0.51	6.45	99.04	72.84±4.69	6.44	91.04	758.20±25.70	3.39	94.78
−80 °C for 1 month	7.32±0.15	2.04	91.45	74.28±3.68	4.96	92.85	787.46±25.13	3.19	98.43
3 freeze–thaw cycles at −20 °C	7.58±0.49	6.48	94.84	76.84±1.98	2.58	96.06	742.40±36.68	4.94	92.80

Table 4 The Inter- and Intra-Batch Precision of Analytical Analysis Transfer (Mean±SD, n=5)

Preparation Concentration (ng/mL)	Intra-Day Precision			Inter-Day Precision
	Measured Concentration (ng/mL)	RSD (%)	Accuracy (%)	Measured Concentration (ng/mL)	RSD (%)	Accuracy (%)
8	8.69±0.59	6.74	108.94	8.23±0.61	7.47	103.20
80	80.45±3.24	4.03	100.56	81.66±3.60	4.41	102.08
800	770.64±48.21	6.26	96.33	792.54±37.03	4.67	99.07

Figure 3 Mean plasma concentration distribution profile of MTX in adults and children patients after MTX infusion; the number on the histogram represents sample size at the corresponding blood sampling time point. Inset: 44–96 h plasma concentrations presentation.

Figure 4 Plasma concentration–time profiles of MTX in two patients with delayed MTX elimination after MTX infusion. Inset: plasma concentrations below 1 μmol/L presentation.

Figure 5 Serum creatinine–time profiles of MTX in two patients with delayed MTX elimination after MTX infusion.