Spatial quantitation of antibiotics in bone tissue compartments by laser-capture microdissection coupled with UHPLC-tandem mass spectrometry

Abstract

Bones are the site of multiple diseases requiring chemotherapy, including cancer, arthritis, osteoporosis and infections. Yet limited methodologies are available to investigate the spatial distribution and quantitation of small molecule drugs in bone compartments, due to the difficulty of sectioning undecalcified bones and the interference of decalcification methods with spatially resolved drug quantitation. To measure drug concentrations in distinct anatomical bone regions, we have developed a workflow that enables spatial quantitation of thin undecalcified bone sections by laser-capture microdissection coupled to HPLC/tandem mass spectrometry, and spatial mapping on adjacent sections by mass spectrometry imaging. The adhesive film and staining methods were optimized to facilitate histology staining on the same sections used for mass spectrometry image acquisition, revealing drug accumulation in the underlying bone tissue architecture, for the first time. Absolute spatial concentrations of rifampicin, bedaquiline, doxycycline, vancomycin and several of their active metabolites are shown for both small rodent bones and larger rabbit bones that more closely resemble human bone density. Overlaid MALDI mass spectrometry images of drugs and histology staining enabled the generation of semi-quantitative data from regions of interest within anatomical bone compartments. These data correlated with absolute drug concentrations determined by HPLC-MS/MS in laser-capture microdissection samples. Collectively, these techniques enable semi- and fully quantitative drug distribution investigations within bone tissue compartments for the first time. Our workflow can be translated to image and quantify not only drugs but also biomarkers of disease to investigate drug penetration as well as mechanisms underlying bone disorders.

Keywords: Antibiotics; Bone penetration; Laser-capture microdissection; MALDI mass spectrometry imaging; Spatial quantitation.

Fig. 1

Workflow schematic from sample preparation to (i) spatial quantitation by laser-capture microdissection (LCM) coupled to UHPLC/tandem mass spectrometry (LC–MS) or (ii) MALDI mass spectrometry imaging (MSI) followed by same-section histology staining with hematoxylin and eosin (H&E)

Fig. 2

Histology staining of mouse and rabbit undecalcified bone sections. (a) Optimization of tissue section thickness for high-quality hematoxylin and eosin (H&E) staining of mouse bones (tibia and femur). (b) H&E staining of 10-μm-thick sections of rabbit vertebrae (left) and femur head (right). Scale bars: 2 mm

Fig. 3

Determination of broad-spectrum antibiotic concentrations in mouse bone compartments by laser-capture microdissection (LCM) coupled to LC–MS/MS. (a) Concentration ratios of rifampicin (RIF, n = 6 bones from 5 mice), doxycycline (DOX, N = 2 bones from 2 mice) and vancomycin (VAN, n = 2 bones from 2 mice) in cortical bone (C), red marrow (M), spongy bone (S) and surrounding muscle tissue (Mu) relative to plasma concentrations obtained at the time of necropsy. (b) Schematic showing mouse bone sections pre- and post-LCM and dissected areas for each compartment shown in (a), using matching color coding

Fig. 4

Quantitation of anti-tuberculosis agent bedaquiline and its major active metabolite in rabbit bone compartments by LCM coupled to LC–MS/MS. (a) Schematic showing rabbit femur head and vertebrae sections pre- and post-LCM, including the 5 anatomically distinct bone areas sampled by LCM. (b) Absolute concentrations (top row) and concentration ratios relative to plasma (bottom row) of bedaquiline and desmethyl bedaquiline (BDQ) in cartilage, spongy bone, red marrow, cortical bone and spinal cord. Compartment numbering as described in (a)

Fig. 5

Semi-quantitative imaging of bedaquiline in rabbit femur compartments by MALDI mass spectrometry. (a) Top row: H&E-stained section and corresponding MALDI MS images of bedaquiline and its active metabolite (desmethyl bedaquiline). Lower left: delineation of the three major regions of interest based on H&E staining and MS images: cortical bone, spongy bone and red marrow; MS image of a matrix cluster ion at m/z 656.0529; and a merged ion image of bedaquiline and the matrix ion. (b) Average ion spectra of bedaquiline and desmethyl bedaquiline in the three regions of interest outlined in (a), showing the characteristic signature of the two naturally occurring bromine isotopes (c) Scatter plots of relative pixel intensities within the three regions of interest outlined in (a)