. Author manuscript; available in PMC: 2014 Jan 22.

Published in final edited form as: Clin Pharmacol Ther. 2013 Apr 10;94(1):126–141. doi: 10.1038/clpt.2013.78

Table 3.

Direct and indirect methodologies for the estimation of intracellular drug concentrations

Direct measurement method^a	Analyte/matrix	Detection method	Utility/limitations/assumptions
Capillary electrophoresis^51,52,⁸⁸	“Bioparticles”, whole cells, organelles	Laser-induced fluorescence, UV, electrochemical, LC–MS	Nano- to femtoliter sample volumes required for analysis
			Technically challenging and limited accessibility
			Can isolate individual cells or organelles for analysis
			Multiple techniques for different culture/ cell types
MSI: Nano-SIMS, MIMS^89–91	Individual cells, potentially subcellular fractions, “bioparticles”	Secondary ion mass spectrometry, with mass analyzer, multi-isotope imaging mass spectrometry	Nano-SIMS ¹⁴C resolution potentially <0.1 µm
MSI: Nano-SIMS, MIMS^89–91			Nano-SIMS sensitivity could achieve 1,000 times that of ¹⁴C autoradiography
Raman microscopy^92–94	Analysis of cells and tissues, material surface	Light scatter through change in polarization potential, rotation, or vibration energy	Probes’ vibrational states within chemical bonds
			Applicable to biological systems with lower energy excitation for sample preservation
			Recent, advanced detection systems have shortened data collection times for increased imaging throughput
Nuclear microscopy (microbeam PIXE/PIGE)^53,54,⁹⁵	Single cell; platinum and endogenous metals	Ion microbeam with particle-induced X-ray/γ-ray emission	Achieves ≤1-µm diameter resolution
			Not a widely accessible technology
			Limited to metal-containing drugs/ compounds (e.g., platinum drugs)
Microautoradiography^49,⁹⁶	Radiolabeled sample in cryosection	Exposure of radiolabel, FISH, IHC, confocal microscopy	Grain density evaluation can be combined with micro-FISH and confocal microscopy for structure–function analyses
Microautoradiography^49,⁹⁶	Radiolabeled sample in cryosection	Exposure of radiolabel, FISH, IHC, confocal microscopy	Resolution generally limited to multicellular level
PET/SPECT imaging^45,46,⁹⁷	Positron/γ particle–emitting total drug or metabolite(s) in imaged tissue or organs of interest	PET/SPECT image with PK samples/LC–MS/LSC	Residualizing vs. nonresidualizing isotopes allow for derivation of internalization rate and concentration
			Receptor occupancy measurements possible
			Expensive, technically challenging, limited by resolution to mathematically deriving concentrations in tissues
PET imaging with simultaneous microdialysis⁴⁸	Same as PET plus microdialysate from volume of interest corresponding to PET scan	PET/in-line HPLC radioligand detector, γ-counter	Requires kinetic modeling to parameterize analyte flux and derive intracellular concentrations
			Similar limitations as PET, yields small sample volumes
			Requires physicochemical characterization of the test article to draw meaningful conclusions
Bulk analysis^11,40	Total drug or metabolite(s) in tissue homogenate or section	HPLC–UV, LC–MS, radioactivity, MALDI	Often fails to describe suborgan distribution
			Pharmacokinetic model-based approach often used to derive intracellular concentrations from resultant data
			Low-technology method and easily accessible

FISH, fluorescence in situ hybridization; HPLC, high-performance liquid chromatography; IHC, immunohistochemistry; LC–MS, liquid chromatography–mass spectrometry; LSC, liquid scintillation counting; MALDI, matrix-assisted laser desorption ionization; MIMS, multi-isotope imaging mass spectrometry; MSI, mass spectrometry imaging; PET/SPECT, positron emission tomography/single-photon emission computed tomography; PIGE, particle-induced γ-ray emission; PIXE, particle-induced X-ray emission; PK, pharmacokinetic; SIMS, secondary ion mass spectrometry; UV, ultraviolet.

Refs. 76–98 are listed in Supplementary Data online.