Table 5.
Published LC–MS/MS assays for quantification of immunosuppressive drugs in biopsies from transplanted organs.
| Drug | Subjects | Extraction/Injection volume/Recovery | Correlation blood vs tissue | Chromatographic conditions | Precursor ion (m/z) > production (m/z | Ref. |
|---|---|---|---|---|---|---|
| CsA | RTRs (n = 21) | Tissues solubilized in digestion buffer (Proteinase K solution in a tissue lysis (ATL)buffer) at 56°C/2 h Precipitate with 0.4 M zinc sulfate: MeOH, 20:80 (v:v) | r = 0.168, | Gradient: (A): 50% MeOH, MeOH (B), both contain 2 mM ammonium acetate/0.1% FA | [M + NH4]+ | [143] |
| IV: 25 μl | p = 0.53 | Online cleaning: POROS R1/20 | CsA: 1220.0 > 1203.0 | |||
| R: NR | Column: Luna Phenyl-Hexyl (Phenomenex) | IS: labeled CsA | ||||
| Run time: 5.5 min | ||||||
| LLOQ: 1 ng/ml | ||||||
|
| ||||||
| CsA | Biopsies (n = 19) from 7 HTRs | Tissues homogenized with water and mixed with two parts of ACN:MeOH: H2O (1:1:3, v:v:v). Precipitation with ACN. The organic phase was dried and reconstituted with MPs A:B, 65:35 (v:v) | NR | Gradient: 20% ACN (A), 80% ACN (B), both contain 20 mM ammonium format | [M + H]+ | [118] |
| IV: 20 μl | Column: (Acquity) UPLC C18 (Waters) | CsA: 1203.7 > 1101.7/1185.7 | ||||
| R: NR | Run time: 36 min | IS: CsC | ||||
| LLOQ: 0.25 ng/ml | ||||||
|
| ||||||
| MPA | Biopsies (n = 4) from 4 RTRs | Tissues grounded to a fine powder and reconstituted with PBS (pH 7.4). Add 60 μl of HCl (0.4 M) and 1 ml of tertiary-butyl methyl ether. Evaporate/reconstitute with 1:1 (v:v) MeOH: H2O. | NR | Gradient: H20 (A), and MeOH (B), both containing 2 mM ammonium acetate/0.1% formic acid | [M + H]+. | [145] |
| IV: NR | Column: Luna Phenyl-Hexyl (Phenomenex) | MPA: 321.1 > 207.3 | ||||
| R: 97% | Run time: 2.2 min | IS: N-phthaloyl-l-phenylalanine | ||||
| LLOQ: 0.6 ng/ml | ||||||
|
| ||||||
| TAC | Biopsies (NR) from 90 and 146 LTRs | Tissues homogenized PBS (0.1 mol/l, pH = 6.5 Extracted with MeOH/ethyl acetate, dried and reconstituted with MeOH. | Banff score | Isocratic: 70% MeOH (B) containing 2 mM ammonium acetate/0.1% FA | [M + NH4] + | [123,142] |
| IV: 20 μl | (r2 =0.984, p = 0.002) | Column: C18 cartridge, (Phenomenex) | TAC: 822 > 768 | |||
| R: 75.3-83.1% | Run time: 1.5 min | IS: ASC | ||||
| LLOQ: 5 pg/mg | ||||||
|
| ||||||
| TAC | Biopsies (n = 6) from 2 RTRs | Tissues solubilized in digestion buffer. Mix with 7 μl of IS + 300 μl water+1 ml of tert-butyl methyl ether in glass tube. Organic phase evaporated and reconstituted in 50:50 MeOH: H2O | NR | Gradient: H2O (A) and MeOH (B) both contain 2 mM ammonium acetate/0.1% FA | [M + NH4] + | [144] |
| IV: 25 μl | Column: Luna Phenyl-Hexyl (Phenomenex) | TAC: 821.5 > 768.6 | ||||
| R: 70% | Run time: 2 min | IS: ASC | ||||
| LLOQ: 0.04 ng/ml | ||||||
Ammonium adduct: [M+NH4]+; ACN: Acetonitrile; ASC: Ascomycin; CsA: Cyclosporine A; CsA: Cyclosporine C; EVE: Everolimus; FA: Formic acid; HTRs: Heart transplant recipients: IV: Injection volume: Ion adduct [M + H]+: LTRs: Liver transplant recipients: LLOQ: Lower limit of quantification: MeOH: Methanol: MPA: Mycophenolic acid: NR: Not reported: PBS: Phosphate buffer solution: R: Recovery: RTRs: Renal transplant recipients: SPE: Solid-phase extraction: TAC: Tacrolimus.