Reagent precoated targets for rapid in-tissue derivatization of the anti-tuberculosis drug isoniazid followed by MALDI imaging mass spectrometry

Article abstract of DOI:10.1007/s13361-011-0150-8

Isoniazid (INH) is an important component of front-line anti-tuberculosis therapy with good serum pharmacokinetics but unknown ability to penetrate tuberculous lesions. However, endogenous background interferences hinder our ability to directly analyze INH in tissues. Chemical derivatization has been successfully used to measure isoniazid directly from tissue samples using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). MALDI targets were pretreated with trans-cinnamaldehyde (CA) prior to mounting tissue slices. Isoniazid present in the tissues was efficiently derivatized and the INH-CA product measured by MS/MS. Precoating of MALDI targets allows the tissues to be directly thaw-mounted and derivatized, thus simplifying the preparation. A time-course series of tissues from tuberculosis infected/INH dosed animals were assayed and the MALDI MS/MS response correlates well with the amount of INH determined to be in the tissues by high-performance liquid chromatography (HPLC)-MS/MS.

Full text of DOI:10.1007/s13361-011-0150-8

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2011) 22:1409Y1419
DOI: 10.1007/s13361-011-0150-8
RESEARCH ARTICLE
Reagent Precoated Targets for Rapid In-Tissue
Derivatization of the Anti-Tuberculosis Drug
Isoniazid Followed by MALDI Imaging Mass
Spectrometry
M. Lisa Manier,1 Michelle L. Reyzer,1 Anne Goh,2 Veronique Dartois,2 Laura E. Via,3
Clifton E. Barry III,3 Richard M. Caprioli1,4
1Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee, USA
2Novartis Institute for Tropical Diseases, Biopolis, Singapore
3National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
4Departments of Chemistry, Pharmacology, and Biochemistry, Vanderbilt University School of Medicine, 465 21st Avenue
South, Medical Research Building 3, Room 9160, Nashville, TN 37232–8575, USA
Abstract
Isoniazid (INH) is an important component of front-line anti-tuberculosis therapy with good serum
pharmacokinetics but unknown ability to penetrate tuberculous lesions. However, endogenous
background interferences hinder our ability to directly analyze INH in tissues. Chemical
derivatization has been successfully used to measure isoniazid directly from tissue samples
using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS).
MALDI targets were pretreated with trans-cinnamaldehyde (CA) prior to mounting tissue slices.
Isoniazid present in the tissues was efficiently derivatized and the INH-CA product measured by
MS/MS. Precoating of MALDI targets allows the tissues to be directly thaw-mounted and
derivatized, thus simplifying the preparation. A time-course series of tissues from tuberculosis
infected/INH dosed animals were assayed and the MALDI MS/MS response correlates well with
the amount of INH determined to be in the tissues by high-performance liquid chromatography
(HPLC)-MS/MS.
Key words: Matrix-assisted laser desorption/ionization, Imaging mass spectrometry, Isoniazid,
Tuberculosis, trans-cinnamaldehyde, Derivatization, Precoated MALDI targets
mole/femtomole range for many compounds), and relatively
fast sampling speeds (up to 100 ms/pixel, depending on
desired resolution and sensitivity). Imaging mass spectrom-
Introduction
ince the introduction of matrix-assisted laser desorption/
S
ionization (MALDI)-based imaging mass spectrometry
etry (IMS) allows an assessment of local drug concentrations
in situ within the lesions of infected animals and therefore
adds highly valuable information to simple serum pharma-
cokinetic analyses.
While MALDI IMS is becoming increasingly popular for
the analysis of drugs and metabolites within tissue sections,
it can be challenging due to low molecular weight back-
ground signals. Endogenous species that may produce
spectral interferences and/or contribute to ionization sup-
[1], this technology has been used to study the spatial
localization of a variety of analytes directly from thin tissue
sections, including proteins [2–4], peptides [5, 6], lipids [7–
9], and small xenobiotics [10–12]. The advantages of
imaging with MALDI mass spectrometry are numerous,
including simple sample preparation, high sensitivity (atto-
Correspondence to: Richard Caprioli; e-mail: r.caprioli@vanderbilt.edu
Received: 12 January 2011
Revised: 4 April 2011
Accepted: 6 April 2011
Published online: 13 May 2011

1410
M. L. Manier et al.: Isoniazid in-Tissue Derivatization
pression are routinely removed from tissues using washing
protocols prior to protein analysis [13–16] (for example,
successive washes in graded ethanol solutions remove salts
and most lipids); however, this approach may extract or
delocalize small molecule analytes as well. Other approaches
to overcome these interferences have involved the selective
choice of MALDI matrix [17], the use of high-resolution
FT-MS analysis [18], or MS/MS analysis [9, 11, 12].
In cases where compounds exhibit poor ionization
efficiency, chemical derivatization may be employed to
increase ion yields. Chemical derivatization [19, 20] is
routinely used to improve detection of drugs by a variety of
analytical techniques, including gas chromatography (GC),
high-performance liquid chromatography (HPLC), and
increasingly MALDI MS [21–30]. Recently, this approach
has also been used in conjunction with IMS for direct tissue
analysis [31]. For example, Franck et al. utilized the reactive
(N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxy-
phenyl) phosphonium bromide (TMPP) compound to direct
peptide fragmentation towards the N-terminus for in situ
tryptic peptides [31].
Adding a derivatization agent to tissue sections compli-
cates the sample preparation process. The derivatization
agent may contribute to analyte delocalization, signal
suppression, and may interfere with matrix crystal forma-
tion. Precoating of MALDI targets has been employed as a
strategy to apply matrix to a surface for improved reprodu-
cibility or sensitivity. Vorm et al. used fast evaporation of
solvent from matrix solutions to produce a thin layer of
homogenous microcrystals directly on a MALDI target plate
[32]. Regenerated cellulose has been affixed to MALDI
plates followed by treatment with α-cyano-4-hydroxycin-
namic acid (CHCA) in order to collect continuous effluent
from a capillary electrophoresis unit [33]. Similarly, peptides
from protein digests were resolved using capillary reversed-
phase HPLC and deposited onto CHCA-coated MALDI
targets [34]. These precoated targets facilitated off-line
coupling of HPLC separation with MALDI analysis, allow-
ing improved sensitivity for low abundance peptides.
More recently, entire MALDI targets were precoated with
2,5-dihydroxybenzoic acid matrix using sublimation. Tissue
sections were thaw-mounted onto the precoated targets and
directly analyzed by MALDI IMS. This approach resulted in
1–2 μm sized crystals with minimal analyte delocalization as
no extraction solvents were applied. Lipids, peptides, and
small molecules (drug compounds) were amenable to
analysis using this technique [35].
after discontinuing chemotherapy. The poor sterilizing
activity of the four drug combination is reflected in the 6–
8 mo regimen required to achieve a sterile cure. There are
many different theories around the intrinsic sterilizing
potential of various drugs [39] but perhaps the most
straightforward is related to the ability of drugs to partition
out of serum and into the dense granulomas that form the
basis of much of the lung pathology of the disease. To test
that theory, we have been exploring IMS as a method to
assess the tissue penetration of TB drugs into lesions. Our
initial efforts to study INH distribution within tissue sections
by MALDI IMS proved difficult due to endogenous tissue
interferences. Isoniazid has been derivatized previously
using trans-cinnamaldehyde [40] and N-methyl-9-chloroa-
cridinium triflate [41] to improve its detection by HPLC
with ultraviolet/visible (UV/Vis) analysis. It has also been
analyzed as the copper chelated Schiff base of salicylalde-
hyde-5-sulfonate using capillary zone electrophoresis [42].
Here we present an in-tissue chemical derivatization
protocol for isoniazid in order to attain suitable sensitivity for
imaging analyses of dosed tissue samples using MALDI IMS.
We investigated the derivatization of isoniazid under various
pH, temperature, time, and solvent conditions using several
aldehydes, steroids, and succinimidyl carbamates. The optimal
reagent, trans-cinnamaldehyde, was then applied to MALDI
targets prior to tissue samples and utilized for the analysis of
lung tissue sections from tuberculosis infected rabbits treated
with a multi-drug mixture containing isoniazid. The reagent
precoated targets allow for rapid in-tissue derivatization and
thus provide a convenient platform for imaging MS studies
where derivatization is required.
Experimental
Materials
The following reagents were purchased from Sigma-Aldrich
Chemical Co. (St. Louis, MO, USA) and used for INH
derivatization: trans-cinnamaldehyde, β-phenylcinnamalde-
hyde, α-bromocinnamaldehyde, 4-hydroxy-3-methoxycinna-
maldehyde, 4-nitrocinnamaldehyde, trans-4-(diethylamino)
cinnamaldehyde, hydrocortisone, and (N-succinimidyloxycar-
bonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bro-
mide (TMPP). Additionally, Waters AccQ-Fluor derivatizing
reagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate)
was purchased from Waters Corporation (Milford, MA, USA).
Methanol and acetonitrile (both HPLC grade) were obtained
from Fisher Scientific (Pittsburgh, PA). Trifluoroacetic acid
(TFA), isoniazid and α-cyano-4-hydroxycinnamic acid
(CHCA) were also obtained from Sigma-Aldrich.
Isoniazid (INH) is widely used in first-line treatment for
tuberculosis as part of a four-drug combination therapy. INH
is well absorbed and has good pharmacokinetic properties
that result in a rapid bactericidal activity on organisms
within patients’ sputum [36, 37]. Unfortunately this activity
is limited to the first month or two of treatment, after which
it is thought to have little long-term “sterilizing” activity
[38]. Sterilizing activity is a complex phenomenon related to
the ability of the drug to prevent relapse with active disease
Method Development with Derivatization
Reagents for MALDI IMS Analysis
Chemical derivatives of isoniazid were formed with the nine
different reagents. Parameters were based on those recom-

M. L. Manier et al.: Isoniazid in-Tissue Derivatization
1411
mended in published methods for derivative reactions of
similar compounds [19, 20, 31, 40, 43–45]. For tissue
analysis, derivatization reagents were applied by either spray
coating, sublimation, or spin coating (detailed below). Based
on preliminary data, trans-cinnamaldehyde (CA) was chosen
for analysis of study samples.
trans-cinnamaldehyde. Tissues were analyzed by two differ-
ent methods: (1) for underivatized INH or (2) for INH
derivatized with CA using precoated targets as detailed
below. In addition, a serial section of each tissue was cut and
mounted on a glass slide and stained with hematoxylin and
eosin (H&E) for anatomical visualization.
CHCA was used as the MALDI matrix and was prepared
as a 15 mg/mL solution in 60:40 acetonitrile:water (with
0.1% TFA). CHCA was coated over the entire surface of all
tissues mounted on MALDI plates. This was accomplished
by placing the solution into a thin layer chromatography
(TLC) glass reagent sprayer (Kontes Glass Company, Vine-
land, NJ, USA) and spraying multiple coats at a distance of
~20 cm across the surface of the tissue. One coating cycle
consisted of passing the sprayer two times across the surface
of the tissue and allowing the tissue to dry. This process was
repeated until there was a homogeneous layer of matrix
crystals over the surface of the tissue, usually about 5–8
cycles. The plates were desiccated for at least 15 min prior to
instrumental analysis.
Animal Studies
Experiments utilizing New Zealand White (NZW) rabbits
aerosol infected with M. tuberculosis as previously
described [46] were performed with the approval of the
animal care and use committee (ACUC) of NIH/NIAID
under assurance #A-4149-01 and protocol #LCID-3. Tuber-
culosis-infected rabbits were dosed with rifampin/isoniazid/
pyrazinamide/moxifloxacin at 30/50/125/25 mg/kg adminis-
tered orally once daily for 1 wk, then sacrificed at various
time points after the final dose. Control rabbits were dosed
with rifampin (24 mg/kg). For MALDI analyses, lung
regions containing lesions were snap-frozen in vapor phase
liquid nitrogen, subjected to 3 Mrad γ radiation as a
sterilization procedure, shipped frozen on dry ice, and stored
at −80 °C until use.
Pretreatment of MALDI Plates with CA Using
High Velocity Spin Coating
In order to create precoated targets, trans-cinnamaldehyde was
deposited on MALDI target plates using high velocity spin
coating. A target was vacuum sealed to the chuck of a WS-400
bench-top single wafer spinner (Laurell Technologies Corpo-
ration, North Wales, PA, USA). Approximately 1 mL of 50%
CA in methanol was deposited onto the center of the plate and
spinning commenced for 10 s at 500 rpm for an initial spread of
reagent. The speed was then increased to 2000 rpm for 20 s to
form a homogeneous coating of CA. The CA-coated plate was
stored overnight in the dark at room temperature to allow
crystal formation prior to tissue placement the following day.
After tissues were thaw-mounted, the plates were allowed to
incubate for at least 30 min at room temperature prior to matrix
application as detailed above.
Tissue Preparation for HPLC-MS/MS Analysis
Drug containing tissues were processed at the time of
sacrifice (BSL-3 facility) with the sample preparation
procedure described below, and the final lyophilized extracts
were shipped for quantitative analysis. This preparation
procedure was validated to sterilize the samples prior to
shipment. Tissue was homogenized in phosphate buffered
saline (PBS) using a Qiagen TissueLyser bead beater
(Hilden, North Rhine-Westphalia, Germany), with 1 mL
PBS added for every 0.2 g of tissue. Warfarin was added as
an internal standard. Proteins were precipitated and INH
extracted by addition of acetonitrile with 0.2% acetic acid in
a 9:1 ratio of extractant to tissue homogenate. Samples were
vortexed for 1 min, centrifuged at 1000 g for 10 min, and the
supernatant removed. The extracts were lyophilized, shipped
on dry ice, and frozen at −20 °C until they were
reconstituted in mobile phase and analyzed. Drug free tissue
that was infected with M. tuberculosis and sterilized via γ
irradiation was used as naive material for calibration stand-
ards, quality control samples and blanks. Each was spiked
with warfarin internal standard, then processed by the
procedure used for study samples. Calibration standards
ranged from 10.2 ng/mL to 10.4 μg/mL.
HPLC-MS/MS Analysis
Lyophilized extracts were thawed and reconstituted into 200
μL mobile phase. Ten μL were injected into an HPLC
system (Symbiosis Pharma, Spark Systems Solutions, The
Netherlands) and separated at a flow rate of 1 mL/min on a
Gemini C6-phenyl 4.6 × 150 mm, 5 μm column (Phenom-
enex, Torrance, California, USA) held at 35 °C. A gradient
elution was used, starting with 97% mobile phase A (water
with 0.2% acetic acid) and going to 85% mobile phase B
(methanol with 0.2% acetic acid) in 4.5 min. The analysis
was performed using an Applied Biosystems/Sciex API4000
triple-quadrupole mass spectrometer (Foster City, California,
USA). Selected reaction monitoring (SRM) of precursor-to-
product transitions were used in electrospray positive
ionization mode to track presence of analytes. Quantitation
of drug levels was performed using the m/z transitions
Tissue Preparation for MALDI IMS
The frozen tissues were cut at −20 °C into 12-μm thick
sections on a cryostat (Leica Microsystems Inc., Bannock-
burn, IL, USA) and thaw-mounted directly onto gold-coated
stainless-steel MALDI target plates (Applied Biosystems,
Foster City, CA, USA) or onto target plates precoated with

1412
M. L. Manier et al.: Isoniazid in-Tissue Derivatization
309.2→251.1 for warfarin and 138.0→121.1 for isoniazid
with collision energies of 28 and 21 eV, respectively.
Although rifampin, pyrazinamide, and moxifloxacin were
quantified also, results are not presented here. The flow rates
of curtain and collision gas were set to 25 and 6 psi,
respectively. Ultra pure nitrogen was used with a nebulizing
temperature of 500 °C. The ion spray voltage, nebulizing gas
(GS1) and Turbo gas (GS2) were set at 5500 V, 45 and 60
psi, respectively. The dwell time for each SRM transition
was 100 ms. Compound specific MS parameters were
optimized for each analyte. Calibration curve data were
evaluated using a quadratic fit and 1/x weighting. Sample
analysis was accepted if the low level quality control
samples were within 20% of nominal concentration and
15% for mid and high level quality control samples.
MALDI Imaging Mass Spectrometry
Mass spectra were acquired in positive-ion mode using a
MALDI LTQ XL linear ion trap mass spectrometer (Thermo
N
*
MS/MS of m/z138 →
(a)
121
INH std
121
NH 2
O
N
H
87
138
138
111
CHCA
110
111
120
96
Control lung
Dosed lung
*
121
120
111
95
*
110
121
80
90
100
110
120
130
140
150
m/z
MS/MS image of INH
(m/z138 121)
Optical image of CHCA
coated MALDI plate
(b)
Control
1hr 5 min
Figure 1. Analysis of underivatized INH in rabbit lung tissue. The animal was dosed with rifampin/isoniazid/pyrazinamide/
moxifloxacin at 30/50/125/25 mg/kg/d for 1 wk and sacrificed 1 h 5 min after the final dose. Non-INH dosed control tissue was
also analyzed. (a) MS/MS spectra for INH (m/z 138→) from prepared standard (250 pmol on plate), CHCA blank, control lung,
and dosed lung. (b) Optical image of rabbit tissue sections coated with CHCA (left) and reconstructed ion image of INH in rabbit
tissues (right). The transition (m/z 138→121) was monitored. Due to endogenous background, it is not possible to distinguish
dosed from control rabbit lung

M. L. Manier et al.: Isoniazid in-Tissue Derivatization
1413
Scientific, San Jose, CA, USA), equipped with a 337-nm
nitrogen laser operating at 60 Hz and resulting in ~40 × 100
μm laser spot size. Dissociation of protonated molecules was
achieved using an isolation window of 1 Da and CID energy
of 40 for INH or 50 for INH-CA (arbitrary units) with
helium as the trapping/collision gas. Isolated precursor ions
were activated for 30 ms using an rf frequency activation Q
value of 0.5 (INH) or 0.25 (INH-CA). Product ion mass
spectra were acquired over the m/z range 75–170 (INH) and
65–300 (INH-CA). For MS/MS imaging experiments, an
average of 15 laser shots per scan was used to produce a
mass spectrum every 200 μm laterally across the tissue. Two
dimensional ion density images were extracted from the raw
data using ImageQuest ver. 1.0.1 (Thermo Scientific, San
Jose, CA, USA).
Results and Discussion
Imaging MS/MS Analysis of Underivatized INH
Analysis of INH by mass spectrometry is straightforward
using high-performance liquid chromatography and electro-
spray ionization [47–51]. However, due to endogenous
interfering components at this very low mass range in tissue,
the in situ analysis of INH by MALDI has been challenging.
Fragmentation of underivatized INH yields predominantly
one product ion (m/z 138→121) as shown for the standard in
Figure 1a (top spectrum). Unfortunately, there is also an
endogenous background signal in rabbit lung tissue with the
same transition (Figure 1a, control lung spectrum). Due to
high background at m/z 121 in the lung tissue, it is not
possible to easily distinguish control from dosed (1 h 5 min)
tissue (Figure 1b), even though this tissue contains a
relatively high concentration of INH (approximately 10 μg/
g tissue, as determined by HPLC-MS/MS). MS3 capabilities
of the ion trap (m/z 138→121→) were also utilized but did
not improve the results. Therefore, derivatization of INH in
lung tissue was investigated.
Chemical Derivatization of INH
We explored derivatives of INH in solution with each of the
reagents listed in Table 1 under various solvent, pH,
concentration, temperature, and time conditions following
recommended published methods for similar derivative
reactions [19, 20, 31, 40, 43–45]. Listed are INH-derivative
precursor ions ([M]+ or [M + H]+), predominant MS/MS
fragments, derivative-to-INH molar ratios, and reaction
parameters. Under the conditions used, some reagents
formed derivatives in solution virtually immediately at room
temperature (e.g., trans-cinnamaldehyde) while others were
more easily formed at higher temperatures and elevated pH
conditions (TMPP). The most promising derivatization
agents were further evaluated on tissue sections and were
applied by spray or spin coating or by sublimation [7] as
described previously.

1414
M. L. Manier et al.: Isoniazid in-Tissue Derivatization
Sublimation is an obvious choice to precoat reagent on
performed. trans-Cinnamaldehyde was used for derivatiza-
tion of INH in subsequent tissue analysis because of its ease
of use and improved sensitivity compared with the other
derivatization reagents.
targets because it forms a very homogeneous coating in a
relatively short period of time (5–15 min). While trans-
cinnamaldehyde provided excellent sensitivity in derivatiz-
ing INH, it is a liquid and thus not amenable to sublimation.
In addition, MALDI plates coated with CA via spin coating
required overnight drying prior to use. Thus, other cinna-
maldehyde-based derivatization agents, which are solids at
room temperature, were evaluated including β-phenylcinna-
maldehyde and 4-hydroxy-3-methoxycinnamaldehyde
(Methods section and Table 1). These reagents were
precoated onto MALDI targets using sublimation proce-
dures. Uniform solid coatings of reagents were obtained;
however derivatization of INH in tissue samples did not
occur on the precoated target alone in the absence of added
solvent. Thus, the target plates were placed in a chamber
containing methanol and acetonitrile vapor at room temper-
ature for 10 min to 1 h in order to wet the tissues and
promote INH-derivative formation. Plates were then coated
with CHCA matrix. The reproducibility of the INH-
derivative signal was not reliable plate-to-plate and, in some
instances, no significant signal was obtained above undosed
tissue background; therefore no further work was done with
these reagents.
The structures of INH, CA, and the INH-CA product are
shown in Scheme 1. The carbonyl of cinnamaldehyde forms
a stable Schiff base with the hydrazide moiety of INH
resulting in an INH-CA compound of [M + H]+ = 252.1.
This derivative forms quickly; CA vapor from INH-CA
standards spotted on MALDI plates readily derivatized INH
in nearby target wells. MS/MS of the derivative at m/z 252
produces three main fragment ions at m/z 225, 123, and 80
as shown in Figure 2a. No significant signals were observed
from the matrix or cinnamaldehyde itself at these transitions
(Figure 2b). Thus, chemical background from these com-
pounds is quite low.
Reagent Precoated Targets
CA was initially applied to tissues by two techniques. In
one, tissues mounted on MALDI target plates were spray-
coated with a CA solution (10 mM) using a TLC reagent
sprayer. In this case, the CA contacted the tissue as a liquid,
and several coatings were applied over time. In the second
method, CA was applied to MALDI target plates (via a spin-
coating technique), allowed to dry, and tissue sections were
subsequently thaw-mounted on the CA-coated target.
Although CA is a liquid at room temperature, crystals
formed on the target plates, likely due to partial oxidation to
trans-cinnamic acid [52]. Presumably enough unoxidized
CA remains in the crystals to react with the INH, as the
oxidized form would not be expected to form the derivative.
Nuclear magnetic resonance (NMR) analysis of a dried
coating indicated that approximately 1:1 CA:cinnamic acid
was present. Subsequent studies have shown that a mixture
of ~1:1 trans-cinnamaldehyde:trans-cinnamic acid may be
directly applied to MALDI target plates. These plates readily
produce uniform coatings without overnight drying/oxida-
tion. In addition, this mixture has been applied by spray
coating either manually using an artist airbrush or automati-
cally using a robotic sprayer (TM Sprayer; HTX Technol-
ogies, LLC, Carrboro, NC, USA) with excellent results.
Tissues applied to plates spin-coated with CA generally
gave ~10-fold more INH-CA signal than tissues that were
Hydrocortisone produced an INH derivative ([M + H]+ =
482) with standard INH, however it was not further
evaluated in tissue. The major MS/MS fragments of the
INH-hydrocortisone derivative are m/z 464 and 452, corre-
sponding to losses of H2O and CH2O, respectively. These
are not very specific losses and therefore potentially more
susceptible to endogenous interferences. Two succinimidyl
carbamates were studied, Waters AccQ-Fluor and TMPP,
which both formed detectable derivatives with INH in
solution. Waters AccQ-Fluor was not evaluated in tissue
because the amount of reagent required to coat a target plate
was cost-prohibitive. The other, TMPP, forms a derivative
with INH that is positively charged and thus readily detected
by MS. TMPP was further evaluated with several different
modifiers (Table 1) to obtain pH ~8–9, which is optimal for
derivatization. Better sensitivity was obtained using triethyl-
amine (TEA) versus ammonium hydroxide, borate, or
ammonium bicarbonate buffers. Derivatization of INH in
tissues using TMPP did not appear as promising as trans-
cinnamaldehyde, although extensive optimization was not
N
O
N
H
N
H
+
N
NH2
O
O
N
H
INH
CA
INH-CA
C6H7N3O
C9H8O
C15H13N3O
(M+H)+mi = 138.1
(M+H)+mi = 133.1
(M+H)+mi = 252.1
Scheme 1. Structures of INH, CA, and INH-CA with monoisotopic molecular weights of protonated molecules

M. L. Manier et al.: Isoniazid in-Tissue Derivatization
1415
*
80
100
INH + CA + CHCA Standard
(a)
80
*
*
123
234
225
115
60
40
20
208
137
148
197
107
159
179
0
100
208
(b)
CA + CHCA Blank
234
80
60
40
20
0
157
160
224
180
180
80
100
120
140
200
220
240
260
m/z
Figure 2. MS/MS spectra for INH-CA derivative (m/z 252→) from (a) prepared standard (5 pmol on plate) (b) CA + CHCA blank
spray-coated with the derivatization agent. While it is
difficult to quantify the absolute amount of trans-cinnamal-
dehyde deposited onto the tissues with either method, based
on the weight gained per plate following CA deposition
(approximately 0.06 mg for spray coating and 60 mg for
spin coating), there is approximately 1000× more CA
present on a precoated target, thus providing a greater molar
excess of CA compared to INH.
In order to more accurately determine the molar ratio of
CA needed to effectively derivatize INH in tissues, various
amounts of CA (50 to 5,000,000 pmol) were applied in
discrete areas on dosed tissue sections (calculated to contain
~10 μg INH/g tissue or 70 pmol INH/mg tissue). CHCA
matrix was applied on top of the CA spots. All spots were
evaluated for the INH-CA derivative (m/z 252→80).
Approximately 500,000 pmol is required for maximum
signal (Figure 3). This corresponds to a molar ratio of
roughly 250,000:1 (assuming the tissue section is 1 mg and
roughly 8 × 15 mm = 120 mm2 and the area under the matrix
spot is 3.14 mm2 resulting in ~2 pmol INH/spot). At higher
molar ratios, the INH-CA signals are lower, possibly
because the increased amount of CA alters crystallization
of CHCA or causes signal suppression. For premixed
solutions a molar ratio of CA:INH of ~100 appears to be
optimum (data not shown) suggesting that tissue contains
compounds that may react or interfere with CA derivatiza-
tion of INH. Based on these results, it is likely the precoated
targets contain enough CA to successfully derivatize the
INH present in tissues from dosed animals.
tively, indicating better derivatization with thinner tissue.
However, it is difficult to section unembedded lung tissues
thinly without extensive tearing and folding. Thus, all lung
tissues were sectioned at 12 μm, but because all lung tissues
in this study were processed identically, direct comparison
among these tissues is reasonable.
Imaging MS/MS Analysis of INH-CA in Rabbit
Lung Tissues
A series of lung tissues from rabbits dosed with INH and
sacrificed at various time points, as well as two non-dosed
250,000:1
CA:INH
INH-CA
120
100
80
60
40
20
0
*
0
0.05
0.5
5
50
500 1250 2500 5000
CA added, nmol
Figure 3. The effect of CA:INH ratio on in situ derivatization
of INH. Various amounts of CA (from 0 to 5000 nmol; n = 2–4)
were added to dosed tissue calculated to contain ~70 pmol
INH/mg tissue and the resulting CA derivative (m/z 252→80)
was monitored. The optimum molar ratio was found to be
~250,000:1. Error bars indicate the highest and lowest values
obtained, however, they are too small to be observed for
values of 0–5 nmol
Due to the lack of sufficient INH-dosed lung tissue, we
were unable to conduct additional studies of tissue thickness
versus derivatization. Studies using rabbit liver (3, 6, 12 μm)
showed relative INH-CA intensities of 2, 1.5, 1, respec-

1416
M. L. Manier et al.: Isoniazid in-Tissue Derivatization
123
N
(a)
H
N
MS/MS of m/z 252
→
N
80
O
*
80
*
123
*
225
Std INH-CA
+ CHCA
224
115
224
234
252
208
208
*
80
*
*
123
Dosed lung
234 252
224
Control lung
119
80 100 120 140 160 180 200 220 240 260
m/z
(b)
Optical image of tissue placed on
CA spin-coated plate + CHCA
MS/MS image of INH-CA
(m/z 252 → 80 /TIC)
1:05
Control
1:25
Control
2:30
2:44
3:37
4:58
6:02
16:39
(c)
Figure 4. Analysis of INH-CA derivative in rabbit lung tissue. Animals were dosed with rifampin/isoniazid/pyrazinamide/
moxifloxacin at 30/50/125/25 mg/kg/d for 1 wk and sacrificed at various h:min after final dose. Non-INH dosed control tissues
from two different rabbits were also analyzed. (a) MS/MS spectra for INH-CA derivative (m/z 252→) from prepared standard (125
pmol on plate), dosed lung from the image below (1 h 25 min), and control lung. (b) Optical image and MALDI image of rabbit
tissues. Tissues were placed on targets that had been precoated with a layer of CA followed by CHCA deposition. The
transition (m/z 252→80/total ion current) was used for ion image reconstruction. (c) H&E stained serial section for lung tissue
from the 1 h 5 min rabbit and expanded view of the MALDI image from (b). Lesions apparent as nuclei-rich dark purple areas
(H&E) are indicated by arrows. This protocol allowed visualization of INH in dosed tissues up to ~17 h, with minimal interfering
signal from the control tissue, however no localization to unaffected lung or lesion areas is observed

M. L. Manier et al.: Isoniazid in-Tissue Derivatization
1417
HPLC-MS/MS Lung (ng/g)
MALDI MS/MS Intensity
120
100
80
60
40
20
0
control tissues, were thaw-mounted onto a target plate that was
precoated with CA as described. CHCA matrix was applied
and the tissues were analyzed via MS/MS of m/z 252.
Data were acquired as one file over all of the tissue
sections. There is virtually no endogenous background in
control tissue at the three major fragment masses (m/z
225, 123, and 80) as shown in Figure 4a. The transition
m/z 252→80 was chosen for image reconstruction,
however, reconstructed ion images for m/z 123 and 225
were similar. As shown in Figure 4b, INH is detected in
all of the dosed rabbit tissues up to ~17 h post-dose,
while no significant signal is observed in the non-dosed
control lung tissue. The time-course clearly shows an
accumulation of INH at the early time points followed by
a reduction of signal with time as the drug is cleared from
the body. The Tmax (time to maximum serum concen-
tration) for INH has been found to be 1 h or less and the
t1/2 (the half-life or rate of clearance) is 1 h to 1 h 30 min
in rabbits (Via, Dartois, unpublished).
0
200
400
600
800
1000
Time post-dose (min)
Figure 5. Correlation of MALDI MS/MS and HPLC-MS/MS
data. MALDI signal intensities (m/z 252→80) were obtained by
averaging the intensity (150 scans) over each tissue shown in
Figure 4, while HPLC-MS/MS results are an average of
multiple areas of lung (n = 2–4). For direct comparison, each
value was corrected to % of maximum response. HPLC-MS/
MS variability noted by error bars ( 1σ) while MALDI data is
from a single analysis. A similar trend is observed for both
HPLC-MS/MS and MALDI MS/MS
No significant localization of INH to either the unaffected
lung tissue or lesion area was observed. For example,
Figure 4c shows the H&E stained serial section for lung
tissue from the 1 h 5 min rabbit as well as an expanded view
of the MALDI ion image for the section analyzed in
Figure 4b. Lesions are shown as dark purple regions
(indicated by arrows) and the center one measures 91000
μm. As shown in the MALDI image, the INH-CA product is
uniformly distributed over the tissue and no observable
localization either to visually normal lung or granuloma is
observed. The uniform distribution is not likely a result of
any analyte delocalization; no significant INH-CA signal
appears on the MALDI target outside of the tissue borders.
However, the tissues evaluated in this study did not have
large granulomas, therefore a more thorough assessment
may be necessary using tissues from animals dosed with
high concentrations of INH that also contain a number of
larger lesions.
shown in Figure 4 (average of 150 scans from each tissue
section). For direct comparison, both values were converted
into percent of maximum response. MALDI data were
obtained from tissue located in only one area of the lung
(for example, right upper lobe) while the HPLC data are
averages of multiple lung areas (n = 2–4) and not always
from the same area as that taken for MALDI analysis.
Therefore, some variability is expected (calculated %RSDs
range from 3 to 70 for HPLC lung results). Excised
granulomas were also analyzed by HPLC-MS/MS for INH,
and showed a similar trend (data not shown). Overall, no
major differences in lung versus lesion concentrations of
INH are noted with either HPLC-MS/MS or MALDI IMS.
However, data do show that as the amount of drug in tissue
decreases (as determined by HPLC-MS/MS) the MALDI
MS/MS signal decreases; thus the trends observed for the
two techniques are largely similar.
Correlation of MALDI MS/MS and HPLC-MS/MS
Data
Conclusions
Absolute quantitation of INH in the tissue sections was not
attempted via MALDI MS. The goal of the current study
was to detect INH in tissue sections (via derivatization in
this case) by IMS and evaluate localization of drug in
unaffected lung tissue compared to TB lesion areas.
However, it is reasonable to expect the signal response to
be a function of drug concentration [11, 53]. Therefore, the
MALDI LTQ response for INH-CA from the current rabbit
study was compared with the results obtained by HPLC-MS/
MS. The comparison is shown in Figure 5. Each time point
represents a different animal that was evaluated by both
MALDI and HPLC-MS/MS. The concentration of INH in
unaffected lung tissue as determined by HPLC-MS/MS is
expressed as ng INH/g tissue while the MALDI results are
INH-CA signal intensities (m/z 252→ 80) from tissues
In-tissue derivatization has been shown to improve the
detection of INH in cases where the analysis is hindered by
isobaric interferences. High-resolution analysis may be used
to distinguish isobaric species, however, this capability is
not always available and may not improve the analysis if
sensitivity is also an issue. For these instances, chemical
derivatization is a viable alternative. For low molecular
weight analytes, derivatization should ideally shift the
molecular weight into a less complex spectral region (e.g.,
away from MALDI matrix ions and low mass contaminants).
Sensitivity may be enhanced by using a derivatizing agent
with a fixed charge (e.g., TMPP or Girard’s T). We found,
as anticipated, that the total amount of reagent required for
derivatization on tissue was significantly greater than for an
equivalent amount of analyte in solution. This is due to the

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Acknowledgments
The authors acknowledge support for this work (in part) by
the Bill and Melinda Gates Foundation Accelerator grant
N01 HD23342, (in part) by NIH/NIGMS grant 5R01
GM58008, and (in part) by the Intramural Research Program
of NIAID, NIH. The assistance of Dr. Joey C. Latham, Dr.
Kenneth E. Schriver, and Jamie Allen (Vanderbilt) and
Danielle Weiner and Jacqueline Gonzales (NIH) is gratefully
acknowledged.
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