Table 4.
Published LC–MS/MS assays for quantification of immunosuppressive drugs in lymphocytes.
| Drug | Subjects | Extraction | Chromatographic conditions | Precursor ion (m/z) > product ion (m/z) | Ref. |
|---|---|---|---|---|---|
| CsA | HV (n = NR); LTRs, LgTRs, and RTRs (n = 64) | At 4°C to prevent efflux of CsA | Gradient: H2O (A), MeOH (B) | LC-MS | [110] |
| Lymphocyte isolated from 8 ml blood with BD Vacutainer® CPT™ Cell Preparation Tubes | Online SPE: XTerra™ MS C8 | [M + Na] + | [111] | ||
| Cells lysed with MeOH/dry reconstituted with MeOH | Column: XTerra™ MS C18 (Waters) | CsA: 1224.7/NR | [120] | ||
| IV: 40μl | Run time: 31 min | IS: 27 demethoxy-sirolimus | |||
| R: 98% | LLOQ: 5ng/ml;0.5fg/PBMC | ||||
|
| |||||
| CsA | Early RTRs (n = 20); HTRs (n = 10) | Verapamil added prevent efflux of CsA | Gradient: ACN/20 mM ammonium formate buffer pH 3.6 (20:80) (A), ACN/(NH4 + COO-) pH 3.6 (80:20) (B) | [M + H] + | [118] |
| T-lymphocyte isolated from 7 ml blood with Prepacyte®. | SPE: Water Oasis®, HLB 1 cc, 30 mg | CsA: 1203.7 > 1101.7/1185.7 | [121] | ||
| Cells were lysed and protein precipitated with MeOH: ACN: water (1:1:3) followed by SPE | Column: C8, (Thermo Electron Corp) | IS: CsC | |||
| IV: 100 μl | Run time: 38 min | ||||
| R: CsA and metabolites 73.5–98.6 | LLOQ: 0.25 ng/ml | ||||
|
| |||||
| CsA and metabolites | Early (n = 57) and chronic RTRs (n = 54) | Verapamil added to prevent efflux of CsA | Gradient: 5% ACN (A), 95% ACN (B); both contain 2 mM/l ammonium acetate/0.1 FA | [M + H] + | [122] |
| Lymphocyte isolated from 1.5 ml blood with Histopaque 1077 solution | Column: Acquity UPLC RP BEH, C18 | CsA: 1202.8 > 156.2. | |||
| Precipitation solution ACN: MeOH (40:60, v/v); (v:v:v) | Run time: 5 min | IS: CsD | |||
| IV: NR, | LLOQ: 5 ng/ml | ||||
| R: CsA and metabolites 71.9–78.4%. | |||||
|
| |||||
| CsA | HTRs (n = 17) | 10 ml whole blood incubated with E-Rosette solution, followed by density separation using Histiopaque solution. | Isocratic: ACN: 2 mM ammonium acetate: FA (70:30:0.1) | [M + NH] + | [125] |
| IV: 1 μl | Column: Supelco LC-CN | CsA: 1,220.8 > 1202.7 | |||
| R: NR | Run time: NA | IS: CsD | |||
| LLOQ: 1 pg | |||||
|
| |||||
| EVE | HTRs (n = 36) | At 4°C to prevent efflux of EVE | Gradient: H2O (A), MeOH (B); both contain 2 mM ammonium acetate/0.1% FA | [M + NH4] + | [132] |
| Lymphocyte isolated from 8 ml blood with BD Vacutainer® CPT™ Cell Preparation Tubes. Cells were lysed with MeOH dry & reconstituted with MeOH. Extract one part with 4:5:3 parts of zinc sulfate 0.1 M: 5: H20: CAN | Column: MassTrak TDM C18 (Waters). | EVE: m/z 975.5 > 908.5 | |||
| IV: 20 μl | Run time: 1.5 min | IS: ASC | |||
| R: 79.4–87.1%. | LLOQ: 1.25 ng/ml | ||||
|
| |||||
| TAC | RTRs (n = 65) | At 4°C to prevent efflux of TAC | Isocratic: 90% ACN contains 2 mM ammonium acetate/0.1% FA | [M + NH4] + | [123,124] |
| Lymphocyte isolated from 7 ml blood with Ficoll-Paque Plus solution. Reconstituted with PBS and extracted with 1-chlorobutan. Organic phase dried and reconstituted with MeOH containing 2 mmol/l ammonium acetate/0.1 % FA | Column: Xterra C18 (Waters) | TAC: m/z 821.6 > 768.5 | |||
| IV: 25 μl | Run time: NR | IS: ASC | |||
| R: 74.8–86.7% | LLOQ: 0.01 ng/ml/0.006 ng/106 PBMCs | ||||
|
| |||||
| TAC | HTRs (n = 24) | At 4°C to prevent efflux of TAC | Gradient: H2O (A); MeOH (B) both contain 2 mM ammonium acetate/0.1% FA | NR | [119] |
| Lymphocyte isolated from 7 ml blood with BD Vacutainer® CPT™ Cell Preparation Tubes. Cells lysed with MeOH dried/reconstituted with MeOH and extracted with zinc sulfate 0.1 M: 1: 2.5 (v:v) | Column: MassTrak TDM C18 (Waters) | ||||
| IV: 20 μl | Run time: NR | ||||
| R: 97.2–103.4%. | LLOQ: 12.5 pg/million PBMCs | ||||
Ammonium adduct: [M+NH4]+; ASC: Ascomycin; CsA: Cyclosporine A; CsA: CsC: Cyclosporine C; CsD: Cyclosporine; EVE: Everolimus; FA: Formic acid; HTR: Heart transplant recipient: Ion adduct [M + H7rsqb;+; IV: Injection volume: LTR: Liver transplant recipient: LgTR: Lung transplant recipient: MeOH: Methanol: NR: Not reported: PBS: Phosphate buffer solution: R: Recovery: RTR: Renal transplant recipient; TAC: Tacrolimus.