Table 1.
High performance liquid chromatography parameters for the quantification of MTX and its metabolites.
| Matrix | Analyte | Sample preparation | IS | Column | Mobile phase | Flow rate (mL/min) | Detector Temp. (°C) | Detector | LOD | LOQ | Linear range | Recovery (%) | Ref. |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Human plasma | MTX | SPE | DAPA | C18 (150 mm × 4.6 mm, 5 μm) | 90% PBS (50 μmol/mL, pH 5.3) and 10% ACN | 0.5 | 15–20 | Fuorescence detection at 368 a/425 bnm | 11 pg | 2.3 ng/mL | r2 = 0.999 | 81.2– 81.5 |
[37] |
| Plasma | MTX | Precipitation of proteins with ACN | Not used | C18 (3.0 mm × 75 mm, 2.7 μm) | HA/SAB (85.0 mM, pH 4.00) and 11.2% ACN | 0.4 | – | UV–visible at 305 nm | – | 0.10 μM | 0.10–6.0 μM | 89.6 | [38] |
| Human serum | MTX | Remove of proteins by SNS | Ferulic acid | C18 (250 mm × 4.6 mm, 5 μm) | 78% SPB (10 mM, pH 6.40) and 22% MeOH | 1.0 | 35 | UV–visible at 310 nm | 0.006 μg/mL | 0.02 μg/mL | 0.05–10.0 μg/mL | 97.52 | [39] |
| Human serum | MTX | SPE | Not used | C18 (250 mm × 4.6 mm, 5 μm) | 23% MeOH and 77% aqueous solution of H3PO4 0.05% | 1.0 | – | UV–visible at 306 nm | – | – | – | 81.6–86.2 | [40] |
| RBC | MTX-Gls | Enzymatic treatment by PBS with MCE | Not used | C18 (250 mm × 4.6 mm, 5 μm) | 11% ACN, 89% AAB (0.05 M, pH 5.5) and 0.25% H2O2 30% | 0.6 | – | fluorescence detection at 370 a/463 b nm |
10.9 nmol/L | 32.9 nmol/L | 25–400 nmol/L | 70.3–72.8 | [52] |
| Waste water | MTX | SPE | Nifidipen | C8 (250 mm × 4.6 mm, 5 μm) | 50% ACN and 50% H2O containing 0.1% TFA | 1.0 | Fluorescence detection at 367 a/463 bnm |
0.9 μg/mL | 3.0 μg/mL | r2 = 0.996 | 92.3–110.5 | [53] | |
| Human plasma and urine samples | MTX | Remove of proteins by ACN and H2O (1:1) | Not used | C18 (250 mm × 4.6 mm, 5 μm) | 90% PBS (0.15 M, pH 5.0) and 10% ACN | 1.0 | – | Electrochemical | 0.2 μg/L | – | 0.01–20 mg/L | 83–107 | [54] |
| Macromolecular conjugates | MTX | Dissolvation of drug in SB | Not used | Superdex Peptide (150 mm × 4.6 mm, 5 μm) | 0.1 M SB | 0.4 | 22 | UV–visible at 302 nm | 0.2761 μM | 0.9203 μM | 1.204–40.13 μM | 93.18–104.5 | [56] |
| Urine | MTX | SPE | IBP | C18 (250 mm × 4.6 mm, 5 μm) | 60% ACB (10 mM, pH 4) and 40% MeOH | 1.5 | – | UV–visible at 400 nm | 0.03 μg/mL | 0.08 μg/mL | 0.08–10 μg/mL | 84 | [58] |
| Human serum | MTX | SPE | 1,3,7-trimethyluric acid | C18 (250 mm × 4.6 mm, 5 μm) | 89% SAB (50 mM, pH 3.6) and 11% ACN | 1.0 | 30 | UV–visible at 307 nm | 0.003 μM | 0.01 μM | 0.025–5.00 μM | 93.1–98.2 | [59] |
| Drug | MTX | Dissolvation of drug in 5% DMSO (v/v) PBS | Not used | C18 (250 mm × 4.6 mm, 5 μm) | H2O (including 1% isopropyl alcohol and 0.01% HFBA) and ACN (including 1% isopropyl alcohol and 0.01% HFBA) | 2.0 | 20 | UV–visible at 280 nm | 0.03 μM | 0.10 μM | 1.00–200 μM | – | [60] |
| Drug | MTX | Dissolvation of drug in MP | IBP | C8 (250 mm × 4.6 mm, 5 μm) | 70% MeOH and 30% OPA (1.67%) | 1.5 | – | UV–visible at 254 nm | 3.3 ng | 10.9 ng | 1–500 μg/mL | 99.23–100.69 | [68] |
| Pharmaceutical formulation | MTX | Dissolvation of drug in NaOH | Not used | C18 (250 mm × 4.6 mm, 5 μm) | 85% ACN and 15% PBS | 1.0 | – | UV–visible at 340 nm | 0.6 μg/mL | 0.8 μg/mL | 1–6 μg/mL | 99–100.30 | [69] |
| Drug | Impurities in MTX | Dissolvation of drug in DMSO | Not used | C18 (250 mm × 4.6 mm, 5 μm) | SDHP in water (20 mmol/L, pH 3.0) and ACN | 2.2 | 40 | UV–visible at 305 nm | <0.774 μg/mL | <1.03 μg/mL | r2 > 0.9999 | 95.2–103 and 82.7–117 (at LOQ level) | [70] |
| Tablets, injection, SLNs, SLN-gel and LPHNPs | MTX | Dissolvation of drug in NaOH | Not used | C18 (250 mm × 4.6 mm, 5 μm) | 70% KH2PO4 buffer (10 mM, pH 3), 10% ACN and 20% MeOH | 0.6 | 30 | UV–visible at 302 nm | 0.024 μg/mL | 0.074 μg/mL | 0.01–100 μg/mL | >95 | [77] |
| Plasma, brain and liver | MTX | Dissolvation of drug in PTP 0.5% (w/v) | Not used | C18 (150 mm × 4.6 mm, 5 μm) | 85% PBS (0.01 M, pH 3.9) and 15% ACN | 1.0 | – | UV–visible at 307 nm | – | – | – | – | [78] |
| Microparticles | MTX | Dissolvation of drug in an aqueous AA (1 wt%) | 4-Amino AC | C18 (250 mm × 4.6 mm, 5 μm) | 25% MeOH and 75% AAB (0.05 M, pH 6.0) | 1.0 | – | UV–visible at 303 nm | 0.014 μg/mL | 0.047 μg/mL | 0.5–16 μg/mL | 89.5–105.5 | [80] |
| Plasma | MTX | Precipitation of proteins with ACN | Caffeine | C18 (250 mm × 4.6 mm, 5 μm) | 89% PBS (0.01 M, pH 3.9) and 11% ACN | 1.0 | 30 | UV–visible at 303 nm | 4 ng/mL | 5 ng/mL | 10–1000 ng/mL | 93.02–96.93 | [81] |
| Tablet | MTX | Dissolvation of drug in ACN | Not used | C18 (250 mm × 4.6 mm, 5 μm) | 80% H2O (pH 3 with HF) and 20% ACN | 1.0 | 25 | UV–visible at 211 nm | 0.0000279 × 10−05 μg/mL | 0.0000847 × 10−05 μg/mL | 8–60 μg/mL | 99.46–99.92 | [82] |
| Physiological fluids | MTX | Precipitation of proteins with MeOH and PA | Sparfloxacin | C18 (250 mm × 4.6 mm, 5 μm) | 36% MeOH and 64% TFA (0.05%) | 1.0 | 40 | UV–visible at 290 nm | Refer to text | Refer to text | 0.025–1.0 μg/mL | 98.57 (for plasma), 95.84 (for aqueous humor), 98.51 (for vitreous humor) | [83] |
| Polymeric nanocapsules | MTX | Dissolution of drug in MP | Not used | C18 (150 mm × 4.6 mm, 5 μm) | 65% H2O, 30% ACN, 5% THF (pH 3.0) | 0.8 | 40 | UV–visible at 313 nm | – | 2.61μg/mL | 10–50 μg/mL | 96.74–101.23 | [84] |
| Biopharmaceutical products | MTX | Dissolvation of yeastolate powders in H2O | Not used | C4 (250 mm × 4.6 mm, 5 μm) | 0.05% TFA in water and 0.05% TFA in ACN | 1.0 | 40 | UV–visible at 302 nm | 0.02 μM | 0.09 μM | r2 > 0.9996 | 97–105 | [85] |
IS: Internal standard, RBC: red blood cell, AAB: ammonium acetate buffer, ACB: acetate buffer, TEA: triethylamine, PBS: phosphate buffer solution, AA: acetic acid, ACN: acetonitrile, AA/SAB: acetic acid/sodium acetate buffer, SDHP: sodium dihydrogen phosphate, SPB: sodium phosphate buffer, HF: formic acid, THF: tetrahydrofuran, DAPA: 2,4-diaminopteroic acid, OPA: orthophosphoric acid, IBP: ibuprofen, TFA: trifluoroacetic acid, HFBA: heptafluorobutyric acid, 4-AminoAC: 4-aminoacetophenone, MCE: mercaptoethanol, MP: mobile phase, SB: sodium bicarbonate, SNS: silver nitrate solution, PTP: pentasodium triphosphate, PA: perchloric acid, MTX-Gls: MTX polyglutamates.
a
Excitation.
b
Emission.