Table 2.
Liquid chromatographic and MS conditions of LC-MS methods for MTX and its metabolites quantification.
| Sample | Preparation | LC conditions | Interface | Target analyte | m/. of mass transition | IS | LODa | LOQa | Recovery (%) | Ref. |
|---|---|---|---|---|---|---|---|---|---|---|
| Different surfaces | Whatman filter paper | C18 (2.1 mm × 100 mm, 3.5 μm) column. H2O, ACN and HF 1% as mobile phase at a flow rate of 200 μL/min. | ESI/SRM+ | CYT, GEM, MTX, [13C,2H3] MTX, Et Ph, IFO, CP, IRI, DOX, EPI, VNC |
244.0 > 112.3, 264.7 > 112.3, 455.2 > 308.4, 459.2> 312.2, 691.0 > 691.0, (261.1 > 92.3, 140.2, 154.1, 232.9), (261.1 > 92.3, 140.2, 154.1, 232.9), 587.9 > 587.3, (544.6 > 379.2, 397.1), (544.6 > 379.2, 397.1), 413.3 > 353.2 |
[13C,2H3] MTX | 0.1–1 pg/sample | 0.1 ng/cm2 |
99 | [16] |
| RBCs | SPE | C18 (50 mm × 1.00 mm, 4 μm) column. NH4HCO3 (10 mM, pH 7.5) with 5 mM DMHA, ACN with 5 mM DMHA as mobile phase at a flow rate of 200 μL/min |
ESI/MRM+ | MTXPG1, MTXPG2, MTXPG3, MTXPG4, MTXPG5, MTXPG6, MTXPG7 |
455.2 > 308.1, 584.3 > 308.1, 713.3 > 308.1, 842.3 > 308.1, 971.3 > 308.1, 1100.4 > 308.1, 1229.4 > 308.1 |
Aminopterin | 0.8 nM | 2.5 nM | 31.2–47.8 | [41] |
| Human erythrocyte | SPE | Not used | MALDI/SRM+ | MTX, MTXPG2, MTXPG3, MTXPG4, MTXPG5, MTXPG6, MTXPG7, Aminopterin |
455.2 > 308.2, 584.4 > 308.2, 713.4 > 308.2, 842.4 > 308.2, 971.6 > 308.2, 1100.3 > 308.2, 1229.4 > 308.2, 441.2 > 294.2 |
Aminopterin | 1 nmol/L | 10 nmol/L | 62.4–71.3 | [42] |
| DBS | SPE | C18 (150 mm × 2.1 mm, 3 μm) column. ABB (10 mM, pH 7.5) and ACN as mobile phase with flow rate of 0.15 mL/min |
ESI/MRM+ | MTXPG1, MTXPG2, MTXPG3, MTXPG4, MTXPG5 |
455.40 > 175.05, 584.40 > 175.05, 713.40 > 175.05, 842.30 > 175.05, 971.60 > 175.05 |
Not used | 1.6b nM, 1.5c nM |
5b nM, 4.5c nM |
44–72 | [43] |
| Plasma | SPE | Acquity UPLC BEH shield RP (2.1 mm × 150 mm, 1.7 μm) column, 85% ACN and 15% aqueous HF (0.1%) as mobile phase at 40 °C and flow rate of 0.8 mL/min | ESI/MRM+ | MTX, MP, TG, TU |
455.34 > 308.22, 152.89 > 119.00, 168.01 > 151.08, 128.94 > 111.90 |
TU | – | – | 94.28–102.22 | [47] |
| ECs in liquid and solid environmental matrices |
MAE | Refer to text | ESI/MRM+a | – | – | ACE-D4, IBP-D3, BIS A-D16, CAR-13C6, KET-D3, NAP-D3, SER-D3, TAM, ATE-D5 13C2, PRO-D7 15 N, MET-D6, BEZ-D6, MET-13C, AMP-D5, MET-D5, MDMA-D5, MDA-D5, Heroin-D9, codeine-D6, ketamine-D4, cocaine-D3, BEN-D8, EDDP-D3, MOR-D3, COT-D3, COCA-D8, TEM-D5, 1S,2R-(+) ephedrine-D3, MEPH-D3, METH-D9, NOR-D4, estrone (2,4,16,16-D4), estradiol (2,4,16,16-D4), QUE-D8 HEM, CIS-D6, MET-D7, FLU-D5, MIR-D3 |
6.13–9.04 ng/L and 1.64 ng/g | 20.24–29.83 ng/L and 5.42 ng/g | 40–152 | [50] |
| Plasma | LLE | C18 (50 mm × 3 mm, 4.6 μm) column. MeOH, AAB (2 mM, pH 4.0) as mobile phase at a flow rate of 1 mL/min | HESI/SR+ | MTX, DSN, M-4, TOL |
455.0 > 175.0, 488.1 > 401.0, 444.26 > 401.0, 271.1 > 155.0 |
Tolbutamide | – | 1 ng/mL | 79.4–87.2a | [88] |
| Human saliva | SPE | C18 (150 mm × 2 mm, 2.2 μm) column, 0.1% HF and ACN as mobile phase at a flow rate of 0.4 mL/min |
ESI/MRM+ | MTX, Aminopterin |
455.6 > 308.4, 441.2 > 294.0 |
Aminopterin | 1 ng/mL | – | 89–94 | [89] |
| Drug | Dissolvation of drug in DMSO | C18 (250 mm × 4.6 mm, 5 μm) column, 0.2% HF and ACN as mobile phase at a flow rate of 1 mL/min | ESI+ | C19H21N8O5+, C20H23N8O6+, C20H23N8O6+, C15H17N8O+, C20H22N7O6+, C19H21N8O5+, C20H23N8O6+, C22H27N8O6+, C19H21N8O4+, C15H16N7O2+, C21H25N8O5+, C18H19N8O3+, C22H27N8O5+, C22H27N8O5+, C22H27N8O5+, C15H15N6O3+ |
(441.16278 > 441, 294 ), (71.17325 > 471, 308), (471.17319 > 471, 308), (325.15192 > 305, 308), (456.16232 > 456, 309), (441.16269 > 441, 308), (471.17343 > 471, 324), (499.20453 > 499, 308), (425.16794 > 425, 308), (326.13599 > 326, 175), (469.19379 > 469, 308), (395.15720 > 395, 308), (483.20950 > 483, 308), (483.20947 > 483, 308), (483.20953 > 483, 308), (327.11975 > 327, 309) |
Not used | – | – | – | [90] |
| Pharmaceutical formulations and wipe samples |
Dilution with H2O | C18 (2.1 mm × 100 mm, 3.5 μm) column at 15 °C. H2O, ACN and HF 1% as mobile phase at a flow rate of 200 μL/min | ESI/SRM+ | CYT, GEM, MTX, [13C,2H3] MTX, Et Ph, IFO, CP, IRI, DOX, EPI, VNC |
244.0 > 112.3, 264.7 > 112.3, 455.2 > 308.0, 459.2 > 312.2, 691.0 > 691.0, (261.1 > 92.3, 140.2, 154.1, 232.9), (261.1 > 92.3, 140.2, 154.1, 232.9), 587.9 > 587.3, 544.6 > 379.2, 397.1), (544.6 > 379.2, 397.1), 413.3 > 353.2 |
[13C,2H3] MTX | 0.01 ng/mL | 0.25 ng/mL | 85–110 | [92] |
| Stainless steel surface | Whatman filter paper wetted with 20% H2O and 80% MeOH with 0.1% HF | Biphenyl (50 mm × 4.6 mm) column. H2O (pH 2.3 adjusted with HF-AF) and MeOH as mobile phase at a flow rate of 0.6 mL/min | ESI/MRM+ | 5- FU, OXP, CP, PTX, VNC, MTX |
131 > 114, 398 > 306, 261 > 141, 876.3 > 308.1, 825.6 > 765.6, 455.2 > 308.1 |
MTX-methyl-d3, paclitaxel-d5, vincristine-d3 sulfate, cyclophosphamide-d4 |
0.02 ng/mL | 0.06 ng/mL | 102a | [93] |
| Human whole blood | Precipitation with 50% trifluoroacetic acid, and extraction by EtOAc | C18 (2.1 mm × 100 mm, 3 μm) column. 70% AF (20 mM) and 30% ACN (1% HF) as mobile phase at room temperature and flow rate of 0.2 mL/min | ESI/MRM+ | MTX, Doxofylline | 455.2 > 308.2, 267.2 > 181.2 |
Doxofylline | 0.5 ng/mL | 1 ng/mL | 26.2–37.8 | [94] |
| Caco-2 cells | Protein precipitation by ice-cold ACN | C8 (150 mm × 3.9 mm, 5 μm) column. 0.1% HF and ACN as mobile phase at flow rate of 0.5 mL/min | ESI/MRM+ | MTX, MTX-Glu2, MTX-Glu3, MTX-Glu4, MTX-Glu5, Aminopterin |
455.2 > 308.2, 584.1 > 308.2, 713.1 > 308.2, 842.1 > 308.2, 971.1 > 308.2, 441.3 > 294.2 |
Aminopterin | – | 2 nM | 60-79a | [95] |
| Serum | Protein precipitation by MeOH. | C18 (100 mm × 4.6 mm, 3 μm) column. 88% AA 1% and 12% ACN as mobile phase at a flow rate of 0.5 mL/min | ESI/MRM+ | MTX | 455.2 > 308.1 | Pterin | 3.0 nM | 10.0 nM | 100.4 | [96] |
| Human plasma | SPE | (50 mm × 4.6 mm, 5 μm) column. 70% ACN, 15% AF (10 mM) and 15% HF (0.5%) as mobile phase at 40 °C and flow rate of 0.6 mL/min | ESI/MRM+ | MTX, MTX-d3 |
455.3 > 308.2, 458.3 > 311.2 |
MTX-d3 | – | 0.500 ng/mL | 85.6 | [97] |
| Plasma | Protein precipitation by 70% ACN and 30% H2O | C18 (50 mm × 2.1 mm, 3 μm) column. 0.03% AA and ACN as mobile phase at a flow rate of 0.5 mL/min |
ESI/MRM+ | MTX, .–amino ACP |
455.3 > 308.3, 136.1 > 94.4 |
p–amino ACP | – | 0.05 μmol/L | >94 | [99] |
| Plasma | Protein precipitation by addition of 800 μL of cold 16% perchloric acid |
C18 (2.1 mm × 100 mm, 1.7 μm) column, 21% CH3OH and 79% ABC (10 mM, pH 10) with a flow rate of 0.3 mL/min | ESI/SRM+ | MTX, MTXd3 |
455.2 > 308.2, 458.2 > 311.2 |
MTX-d3 | – | 5 nM | 96–102 | [100] |
| Plasma | Protein precipitation with MeOH | C18 (2.1 mm × 50 mm, 1.7 μm) column at 37 °C. MeOH (0.1% HF) and H2O (0.1% HF) as mobile phase with a flow rate of 0.4 mL/min | ESI/MRM+ | MTX, 7-OH-MTX, MTX-d3 |
455.2 > 308.2, 471.2 > 324.1, 458.2 > 311.2 |
MTX-d3 | – | 0.002 μM | 88.6–92.6 | [101] |
| Drug | Dilution with DMSO | CORTECS® T3 (100 mm × 2.1 mm, 1.6 μm) column at 25 °C. 10 mM AA (pH 5.1) and ACN as mobile phase with a flow rate of 0.4 mL/min |
ESI+ | MTX | 459.2 > 454.4 | – | – | – | – | [102] |
| RBCs | Protein precipitation by adding cold 16% perchloric acid |
C18 (2.1 mm × 100 mm, 1.7 μm) column at 35 °C. ABC (10 mM, pH 10) and MeOH as mobile phase at flow rate of 0.3 mL/min | ESI/SRM+ | MTXPG1, MTXPG2, MTXPG3, MTXPG4, MTXPG5 |
455.2 > 308.2, 584.4 > 308.2, 713.4 > 308.2, 842.4 > 308.2, 971.6 > 308.2 |
Stable-isotope labelled | – | 1 nM | 54–98 | [103] |
| Plasma, brain and urine | Protein precipitation with MeOH and ACN | C18 (150 mm × 3 mm, 3.5 μm) column. 70% AA (5 mM) and 30% MeOH as mobile phase with flow rate of 300 μL/min | For MTX and AMP: ESI/SRM+, for 5-FU and 5-BU: ESI/SRM- |
– | – | 5-BUa | – | 15.6 ng/mL (plasma and brain), 78.1 ng/mL (urine) |
24.1–58.1 | [104] |
| Plasma | SPE | C18e (100 mm × 4.6 mm) column. 25% AA (5 mM, pH 5.0) and 75% ACN as mobile phase with a flow-rate of 0.4–0.8 mL/min | ESI/MRM+ | MTX, TFB, Phenacetin |
455.2 > 308.3, 313.2 > 149.2, 180.3 > 110.2 |
Phenacetin | – | 0.49 ng/mL | 84.8–90.7 | [105] |
| Serum | Protein precipitation by MeOH containing HF | Refer to text | ESI/SRM+ | MTX, 7-OH MTX, DAMPA, MTX-d3 |
455.1 > 308.1, 471.2 > 324.2, 326.1 > 175.1, 458.1 > 311.1 |
MTX-d3 | – | 10 nmol/L | 92.4–107.4 | [106] |
| WWTP | On-line SPE | C18 | ESI/SRM−: BEZ, other analytes: ESI/SRM+ | CP, BEZ, ENA, MTX, OR |
261.0 > 140.0, 360.1 > 273.9, 377.2 > 234.1, 455.1 > 308.0, 496.4 > 319.2 |
Not used | 6–16 ng/L | 19–47 ng/L | 55-125a | [108] |
| Urine | SPE | C18 (100 mm × 2.1 mm, 5 μm) column at 20 °C. AF (4 mM, pH 3.2), ACN as mobile phase with a flow rate of 0.3 mL/min | ESI/MRM+ | MTX, MTX-d3, CP, IF, CP-d4 |
455.10 > 308.2, 455.10 > 311.20, 261.08 > 140, 261.08 > 92, 264.90 > 140 |
CP-d4 and MTX-d3 | 10 pg/mL | 20 pg/mL | 78.3a | [109] |
| Hospital environment sites | Washing wipes with suitable solvents | C18 (150 mm × 2 mm, 3 μm) column. 0.05% HF and MeOH as mobile phase at flow rate of 200 μL/min | ESI/MRM−: 5-FU, other compounds: ESI/MRM+ | 5-FU, C-Pt, CYC, CYT, DOX, GEM, IFO, MTX, MIT |
129.0095 > 85.0033 , 372.0518> 355.0252, 261.0321 > 140.0028, 244.0928 > 112.0505, 544.1813 > 397.0918, 264.0790 > 112.0505, 261.0321 > 182.0132, 455.1786 > 308.1254, 335.1350 > 242.0924 |
Not used | – | 1ng/mL | 87.2a | [110] |
| Urine | Protein precipitation by ACN | Hypersil GOLD™ (100 mm × 2.1 mm, 1.9 μm) column at 25 °C. 0.1% HF and ACN with 0.1% HF as mobile phase at a flow rate of 0.3 mL/min | ESI/SRM+ | MTX, 7-OH-MTX, MTX-d3 |
455.1 > 308.1, 471.1 > 324.1, 458.1 > 311.1 |
MTX-d3 | – | 2.5 nM | 104- 126a | [111] |
IS: Internal standard, LC: liquid chromatography, ACN: acetonitrile, HF: formic acid, TU: thiouracil, SPE: solid phase extraction, MAE: microwave assisted extraction, EtOAc: ethyl acetoacetate, p –amino ACP: p-aminoacetophenone, ECs: emerging contaminants, ACE-D4: acetaminophen-D4, IBP-D3: ibuprofen-D3, BIS A-D16: bisphenol A-D16, CAR-13C6: carbamazepine-13C6, KET-D3: ketoprofen-D3, NAP-D3: naproxen-D3, SER-D3: sertraline-D3, TAM 13C2 15N: tamoxifen 13C2 15N, PRO-D7: propranolol-D7, ATE-D5: atenolol-D5, MET-D6: metformin (dimethyl-D6), BEZ-D6: Bezafibrate-D6, MET-13C: Methylparaben-13C, AMP-D5: amphetamine-D5, MET-D5: methamphetamine-D5, codeine-D6, ketamine-D4, cocaine-D3, BEN-D8: benzoylecgonine-D8, EDDP-D3, MOR-D3: morphine-D3, COT-D3: cotinine-D3, COCA-D8: cocaethylene-D8, TEM-D5: temazepam-D5, 1S,2R-(+) ephedrine-D3, MEPH-D3: mephedrone-D3, METH-D9: methadone-D9, NOR-D4: norketamine-D4, estrone (2,4,16,16-D4), estradiol (2,4,16,16-D4), QUE-D8 HEM: quetiapine-D8 hemifumurate, CIS-D6: citalopram-D6, MET-D7: metoprolol-D7, FLU-D5: fluoxetine-D5, MIR-D3: mirtazapine-D3, AF: ammonium formate, MALDI: matrix-assisted laser desorption ionization, a: (+)-MTX, b: (-)-MTX, DMHA: dimethylhexylamine, RBCs: red blood cells, CP-d4: cyclophosphamide-d4, MTX-d3: methotrexate-d3, AA: acetic acid, 5-FU: 5-fluorouracil, C-Pt: carboplatin, CYC: cyclophosphamide, CYT: cytarabine, DOX: doxorubicin, GEM: gemcitabine, IFO: ifosfamide, MIT: mitomycin C, WWTP: wastewater treatment plants, BEZ: bezafibrate, CP: cyclophosphamide, OR: orlistat, ENA: enalapril, ABC: ammonium bicarbonate, TFB: tofacitinib, DBS: dried blood spots, ABB: NH4HCO3 buffer, OXP: oxaliplatin, PTX: paclitaxel, VNC: vincristine, Et Ph: Etoposide phosphate, IRI: Irinotecan, EPI: Epirubicin, LLE: liquid-liquid extraction, AAB: ammonium acetate buffer, DSN: Dasatinib, M-4: N-deshydroxyethyl dasatinib, TOL: tolbutamide, HESI: heated electron spray ionization, 5-BU: 5-bromouracil.
a
Only for MTX.
b
For individual MTXPGs.
c
For MTXPGtotal.