World J Oncol

World Journal of Oncology, ISSN 1920-4531 print, 1920-454X online, Open Access

Article copyright, the authors; Journal compilation copyright, World J Oncol and Elmer Press Inc

Journal website https://www.wjon.org

Original Article

Volume 15, Number 5, October 2024, pages 825-836

Simultaneous Determination of Methotrexate Concentrations in Human Plasma and Cerebrospinal Fluid Using Two-Dimensional Liquid Chromatography: Applications in Primary Central Nervous System Lymphoma

Figure 3. (A) (a) The chromatogram of blank plasma. (b-f) Clinical patient plasma without MTX. (b) Patient 1 drugs: cyclophosphamide, cytarabine, omeprazole, dexamethasone, ondansetron, levocarnitine, methotrexate, and magnesium isoglycyrrhizinate. (c) Patient 2 drugs: vancomycin, caspofungin, spironolactone, lidocaine, reduced glutathione, methylprednisolone, imipenem and cilastatin, voriconazole, and linezolid. (d) Patient 3 drugs: vitamin B6, furosemide, pantoprazole, methotrexate, magnesium isoglycyrrhizinate, vitamin C, tropisetron, rituximab, indomethacin, and promethazine. (e) Patient 4 drugs: cefoperazone and sulbactam, torasemide, pantoprazole, palonosetron, rituximab, promethazine, methotrexate, levoleucovorin calcium, and recombinant human interleukin. (f) Patient 5 drugs: idarubicin hydrochloride, vindesine, sulfate mesna, cyclophosphamide, omeprazole, prednisone acetate, and meropenem. (B) (a) The chromatogram of blank cerebrospinal fluid. (b-f) Clinical patient cerebrospinal fluid without MTX. (b) Patient 1 drugs: cyclophosphamide, cytarabine, omeprazole, dexamethasone, ondansetron, levocarnitine, methotrexate, and magnesium isoglycyrrhizinate. (c) Patient 2 drugs: vancomycin, caspofungin, spironolactone, lidocaine, reduced glutathione, methylprednisolone, imipenem and cilastatin, voriconazole, and linezolid. (d) Patient 3 drugs: vitamin B6, furosemide, pantoprazole, methotrexate, magnesium isoglycyrrhizinate, vitamin C, tropisetron, rituximab, indomethacin, and promethazine. (e) Patient 4 drugs: cefoperazone and sulbactam, torasemide, pantoprazole, palonosetron, rituximab, promethazine, methotrexate, levoleucovorin calcium, and recombinant human interleukin. (f) Patient 5 drugs: idarubicin hydrochloride, vindesine, sulfate mesna, cyclophosphamide, omeprazole, prednisone acetate, and meropenem. MTX: methotrexate.

Figure 4. (a) The linearity of the standard curve was observed at MTX concentrations that ranged 0.07 to 2.38 µmol/L (cerebrospinal fluid). (b) The linearity of the standard curve was observed at MTX concentrations that ranged 1.10 to 5.51 µmol/L (human plasma). (c) The linearity of the standard curve was observed at MTX concentrations that ranged 0.07 to 5.51 µmol/L (cerebrospinal fluid and human plasma). MTX: methotrexate.

**Table 1.** Intra- and Inter-Assay Precision and Accuracy Results of MTX (n = 5)
	QC levels (µmol/L)	Intra-day	Inter-day
MTX: methotrexate; QC: quality control.
Human plasma	0.07	-0.46	4.93	0.26	0.60
	0.55	-0.33	1.34	0.33	3.79
	1.38	1.10	3.25	-1.33	3.78
	4.13	0.60	1.93	0.66	2.31
Cerebrospinal fluid	0.05	1.32	2.72	0.47	0.67
	0.24	0.36	0.49	1.33	2.82
	0.71	2.17	4.05	2.30	3.56
	1.49	1.60	0.36	1.48	2.42

Table 1. Intra- and Inter-Assay Precision and Accuracy Results of MTX (n = 5)

QC levels (µmol/L)

Intra-day

Inter-day

Accuracy (%)

Precision (%)

Accuracy (%)

Precision (%)

MTX: methotrexate; QC: quality control.

Human plasma

0.07

-0.46

4.93

0.26

0.60

0.55

-0.33

1.34

0.33

3.79

1.38

1.10

3.25

-1.33

3.78

4.13

0.60

1.93

0.66

2.31

Cerebrospinal fluid

0.05

1.32

2.72

0.47

0.67

0.24

0.36

0.49

1.33

2.82

0.71

2.17

4.05

2.30

3.56

1.49

1.60

0.36

1.48

2.42

**Table 2.** Recovery Results of MTX
	QC levels (µmol/L)	Recovery
MTX: methotrexate; QC: quality control; RSD: relative standard deviation; SD: standard deviation.
Human plasma	0.55	99.33 ± 2.12	3.88
	1.38	100.55 ± 2.70	1.95
	4.13	99.99 ± 2.90	0.7
Cerebrospinal fluid	0.24	100.69 ± 4.04	1.67
	0.71	100.85 ± 1.61	2.25
	1.49	99.84 ± 1.46	3.61

Table 2. Recovery Results of MTX

QC levels (µmol/L)

Recovery

Mean ± SD (%)

RSD (%)

MTX: methotrexate; QC: quality control; RSD: relative standard deviation; SD: standard deviation.

Human plasma

0.55

99.33 ± 2.12

3.88

1.38

100.55 ± 2.70

1.95

4.13

99.99 ± 2.90

0.7

Cerebrospinal fluid

0.24

100.69 ± 4.04

1.67

0.71

100.85 ± 1.61

2.25

1.49

99.84 ± 1.46

3.61

**Table 3.** Results of Stability (n = 5)
	Condition	QC levels (µmol/L)	Mean ± SD (%)	RSD (%)
QC: quality control; RSD: relative standard deviation; SD: standard deviation.
Human plasma	Freeze-thaw stability (three cycles)	0.55	98.12 ± 1.70	3.16
		1.38	99.47 ± 1.74	1.27
		4.13	100.89 ± 4.16	1
	Short-term stability (6 h at room temperature)	0.55	99.76 ± 0.74	1.34
		1.38	99.04 ± 4.73	3.46
		4.13	99.86 ± 1.55	0.37
	Long-term stability 30 days at -80 °C)	0.55	100.54 ± 1.57	2.84
		1.38	100.74 ± 2.95	2.12
		4.13	99.80 ± 0.35	0.85
Cerebrospinal fluid	Freeze-thaw stability (three cycles)	0.24	99.31 ± 0.42	1.75
		0.71	100.47 ± 1.42	1.99
		1.49	100.33 ± 0.87	0.66
	Short-term stability (6 h at room temperature)	0.24	98.75 ± 0.85	3.61
		0.71	99.15 ± 1.85	2.63
		1.49	100.56 ± 2.00	2.46
	Long-term stability (30 days at -80 °C)	0.24	99.17 ± 0.30	1.26
		0.71	100.09 ± 1.17	1.64
		1.49	100.83 ± 0.47	0.31

Table 3. Results of Stability (n = 5)

Condition

QC levels (µmol/L)

Mean ± SD (%)

RSD (%)

QC: quality control; RSD: relative standard deviation; SD: standard deviation.

Human plasma

Freeze-thaw stability (three cycles)

0.55

98.12 ± 1.70

3.16

1.38

99.47 ± 1.74

1.27

4.13

100.89 ± 4.16

Short-term stability (6 h at room temperature)

0.55

99.76 ± 0.74

1.34

1.38

99.04 ± 4.73

3.46

4.13

99.86 ± 1.55

0.37

Long-term stability 30 days at -80 °C)

0.55

100.54 ± 1.57

2.84

1.38

100.74 ± 2.95

2.12

4.13

99.80 ± 0.35

0.85

Cerebrospinal fluid

Freeze-thaw stability (three cycles)

0.24

99.31 ± 0.42

1.75

0.71

100.47 ± 1.42

1.99

1.49

100.33 ± 0.87

0.66

Short-term stability (6 h at room temperature)

0.24

98.75 ± 0.85

3.61

0.71

99.15 ± 1.85

2.63

1.49

100.56 ± 2.00

2.46

Long-term stability (30 days at -80 °C)

0.24

99.17 ± 0.30

1.26

0.71

100.09 ± 1.17

1.64

1.49

100.83 ± 0.47

0.31

**Table 4.** Test Results of Plasma and Cerebrospinal Fluid Concentration of Subjects
No.	Mode of administration	Sex	Age (years)	Weight (kg)	Fluid concentration (µmol/L)	Plasma (µmol/L)	Combined with intravenous mannitol
^a24 h. ^b48 h. ^c72 h.
1	6.5 g	Male	59	67.5	1.275	299.644	No
2	5 g	Male	62	69	4.306	155.573	No
3	6.5 g	Male	59	69	3.310	171.422	No
4	3.5 g	Female	45	70	0.848	77.380	Yes
5	3.5 g	Male	72	65	2.101	87.899	No
6	6 g	Female	46	66	1.403	88.177	Yes
7	6 g	Male	50	80	1.162	152.761	Yes
8	5.16 g	Male	66	60	3.961	223.446	Yes
9	6 g	Male	72	66	2.394	145.875	No
10	6 g	Male	50	85	1.420	120.460	Yes
11	6 g	Female	46	66	1.793	161.457	No
12	6.5 g	Male	59	69	3.983	12.554	Yes
13	3.5 g	Male	62	69.5	2.168	129.279	No
14	6 g	Male	72	66	2.109	158.315	No
15	6 g	Male	50	88	1.943	108.490	Yes
16	4.5 g	Female	46	65	1.543	151.782	No
17	6 g	Male	50	88	3.089	103.449	Yes
18	4.7 g	Female	70	60	1.703	122.792	Yes
19	5 g	Male	68	59	2.668	209.483	No
20	4 g	Male	52	58	1.307	42.774	Yes
21	6 g	Male	55	90	2.580	89.987	No
22	1.7 g	Female	68	67.5	0.946	35.847	No
23	6 g	Male	72	66	2.617	148.489	Yes
24	3 g	Male	52	57	0.728	43.607	No
25	6 g	Male	55	90	3.743	77.833	Yes
26	5 g	Male	61	68	3.057	4.967^a	No
27	3 g	Female	48	47	0.512	0.436^a	Yes
28	5.7 g	Female	35	56	0.097^a	3.295^a	No
29	3.5 g	Male	61	65	0.611	2.169^a	No
30	5.7 g	Female	35	54	1.309	0.267^b	Yes
31	3.5 g	Male	61	65	0.037	1.863^a	No
32	6.4 g	Male	52	64	1.491	0.991^a	Yes
33	1.8 g	Male	32	63	0.358	1.461^c	No
34	3.5 g	Female	42	57	0.526	0.099^c	Yes
35	1.5 g	Female	57	60	1.975	0.042^c	No

Table 4. Test Results of Plasma and Cerebrospinal Fluid Concentration of Subjects

No.

Mode of administration

Sex

Age (years)

Weight (kg)

Fluid concentration (µmol/L)

Plasma (µmol/L)

Combined with intravenous mannitol

^a24 h. ^b48 h. ^c72 h.

6.5 g

Male

67.5

1.275

299.644

5 g

Male

4.306

155.573

6.5 g

Male

3.310

171.422

3.5 g

Female

0.848

77.380

Yes

3.5 g

Male

2.101

87.899

6 g

Female

1.403

88.177

Yes

6 g

Male

1.162

152.761

Yes

5.16 g

Male

3.961

223.446

Yes

6 g

Male

2.394

145.875

6 g

Male

1.420

120.460

Yes

6 g

Female

1.793

161.457

6.5 g

Male

3.983

12.554

Yes

3.5 g

Male

69.5

2.168

129.279

6 g

Male

2.109

158.315

6 g

Male

1.943

108.490

Yes

4.5 g

Female

1.543

151.782

6 g

Male

3.089

103.449

Yes

4.7 g

Female

1.703

122.792

Yes

5 g

Male

2.668

209.483

4 g

Male

1.307

42.774

Yes

6 g

Male

2.580

89.987

1.7 g

Female

67.5

0.946

35.847

6 g

Male

2.617

148.489

Yes

3 g

Male

0.728

43.607

6 g

Male

3.743

77.833

Yes

5 g

Male

3.057

4.967^a

3 g

Female

0.512

0.436^a

Yes

5.7 g

Female

0.097^a

3.295^a

3.5 g

Male

0.611

2.169^a

5.7 g

Female

1.309

0.267^b

Yes

3.5 g

Male

0.037

1.863^a

6.4 g

Male

1.491

0.991^a

Yes

1.8 g

Male

0.358

1.461^c

3.5 g

Female

0.526

0.099^c

Yes

1.5 g

Female

1.975

0.042^c