TABLE 1.

Comparison of this study with several previously published analytical methods for SRL.

Study	Method	Internal standard	Blood volume (μl)	Sample preparation	Elution	Column	Mobile phase	Linearity range (ng/ml)	Analytical time (min)	Accuracy (%)
Salm, Taylor et al. (2000)	HPLC-MS	32-O-desmethoxysirolimus	500	PPT by ACN and ZnSO4, followed by SPE	Isocratic	Novapak C18 column (150 mm × 2.1 mm, 4 μm)	80% MeOH, 20% 50 mM NH4AC, pH 5.1	0.2–100	10	94.4–104.4
Zochowska, Bartlomiejczyk et al. (2006)	HPLC-UV	Desmethoxyrapamycin	1500	PPT and extracted with 1-chlorobutan	Isocratic	Supelco RP C16-Amide column (150 mm × 4.6 mm, 5 μm)	60% ACN in water	3–50	15	NR
O'Halloran and Ilett (2008)	LC-MS/MS	Desmethoxyrapamycin Sirolimus-d3	15	PPT by ACN and ZnSO4	Gradient	Supelco C18 column (250 mm × 4.6 mm; 5 µm)	1 ml/L FA and 2 mM NH4AC in MeOH and water	1–50	2.5	NR
Morgan, Brown et al. (2014)	LC-MS/MS	13C2D4-everolimus	20	PPT by ACN and NH4HCO3 and ZnSO4	Gradient	Waters Symmetry C18 column (50 mm × 2.1 mm, 3.5 μm)	2 mM NH4AC and 0.1% FA in MeOH and water	1–49	6	90.7–113.16
Shi et al. (2016)	PS-MS/MS	Sirolimus-d3	200	PPT by MeOH and dried	NR	NR	NR	LLOQ: 2	NR	NR
This study	LC-MS/MS	Sirolimus-d3	100	PPT by MeOH	Gradient	Kinetex C18 column (50 mm × 2.1 mm, 1.7 μm)	0.1 mM FA and 0.05 mM NH4AC in MeOH and water	0.5–50	3	88.7–111.8

Abbreviations: HPLC-MS, high-performance liquid chromatography–mass spectrometry; HPLC-UV, high-performance liquid chromatography–ultraviolet; LC-MS/MS, high-performance liquid chromatography–tandem mass spectrometry; PS-MS/MS, paper spray–tandem mass spectrometry; PPT, protein precipitation; ACN, acetonitrile; ZnSO₄, zinc sulfate; NH₄HCO₃, ammonium bicarbonate; MeOH: methanol; SPE, solid-phase extraction; NR, not reported; C18, octadecyl carbon chain; NH₄AC, ammonium acetate; FA, formic acid; LLOQ, lower limit of quantitation.