Basic ResearchTechnology

Qualitative Analysis of Precipitate Formation on the Surface

and in the Tubules of Dentin Irrigated with Sodium
Hypochlorite and a Final Rinse of Chlorhexidine or QMiX
Kamil P. Kolosowski, HBSc, DDS,* Rana N.S. Sodhi, BSc, MSc, PhD, Anil Kishen, BDS, MDS, PhD,*
and Bettina R. Basrani, DDS, MSc, PhD*
Abstract
Introduction: Interaction of sodium hypochlorite
(NaOCl) mixed with chlorhexidine (CHX) produces a
brown precipitate containing para-chloroaniline (PCA).
When QMiX is mixed with NaOCl, no precipitate forms,
but color change occurs. The aim of this study was to
qualitatively assess the formation of precipitate and
PCA on the surface and in the tubules of dentin irrigated
with NaOCl, followed either by EDTA, NaOCl, and CHX
or by saline and QMiX by using time-of-flight secondary
ion mass spectrometry (TOF-SIMS). Methods: Dentin
blocks were obtained from human maxillary molars,
embedded in resin, and cross-sectioned to expose
dentin. Specimens in group 1 were immersed in 2.5%
NaOCl, followed by 17% EDTA, 2.5% NaOCl, and 2%
CHX. Specimens in group 2 were immersed in 2.5%
NaOCl, followed by saline and QMiX. The dentin surfaces were subjected to TOF-SIMS spectra analysis. Longitudinal sections of dentin blocks were then exposed
and subjected to TOF-SIMS analysis. All samples and
analysis were performed in triplicate for confirmation.
Results: TOF-SIMS analysis of group 1 revealed an
irregular precipitate, containing PCA and CHX breakdown products, on the dentin surfaces, occluding and
extending into the tubules. In TOF-SIMS analysis of
group 2, no precipitates, including PCA, were detected
on the dentin surface or in the tubules. Conclusions:
Within the limitations of this study, precipitate containing PCA was formed in the tubules of dentin irrigated
with NaOCl followed by CHX. No precipitates or PCA
were detected in the tubules of dentin irrigated with
NaOCl followed by saline and QMiX. (J Endod
2014;40:20362040)

Key Words
Chlorhexidine, hypochlorite, parachloroaniline, TOFSIMS, tubules

icrobial infections are the most common cause of an endodontic disease (1). To
critically reduce bacterial loads, endodontic treatment regimens combine mechanical instrumentation with chemicals, namely irrigation and medication (2, 3).
The most commonly used irrigation solution is sodium hypochlorite (NaOCl). It is
antimicrobial and an effective tissue-dissolving agent (46). It is used at a
concentration varying between 0.5% and 6% (4). One disadvantage of NaOCl is its ineffectiveness in removing the smear layer (7). Chlorhexidine digluconate (CHX) has also
been suggested as a possible irrigation solution in endodontic treatment (4, 6). It
exhibits antimicrobial activity in addition to substantivity (4, 6, 8). The
concentrations used vary from 0.12% to 2% (4, 6). Its mode of action is a cationic
binding, which causes bacterial cell membrane disruption (9). As a disadvantage,
CHX is ineffective in removing the smear layer and possesses no tissue-dissolving ability
(10). EDTA is a chelating agent that dissolves inorganic components of the dentin but
not the organic components (4, 6, 7). Concentration used is normally 17% (4).
Although its main function is to remove smear layer, dentin erosion can occur with prolonged exposures of 10 minutes (4, 11). QMiX is a novel irrigation compound
containing EDTA, CHX, and a nonspecified detergent (12). It is antimicrobial and
has been shown to remove smear layer. The detergents function is to decrease surface
tension and increase surface wettability (12, 13).
In vitro studies showed that NaOCl and CHX can diffuse into dentinal tubules
(DTs) to a depth of 300 mm (14, 15). Interestingly, the effect of QMiX on bacteria
in 2 studies was reported to extend deeper into the tubules than CHX and similar to
NaOCl, up to 500 mm (16, 17). In endodontics, irrigation solutions are used in
succession, and it is crucial to discern that irrigation solutions used in succession
can react with each other. This is especially evident when NaOCl is mixed with CHX.
This interaction breaks down CHX, produces a brown precipitate containing parachloroaniline (PCA) (1820), and can occlude DTs (21). The amount of PCA is directly
proportional to the concentration of NaOCl used (18). A reaction also occurs when
QMiX is mixed with NaOCl, resulting in a visually detectable color change but without
precipitate formation (pilot study).
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a method used in
surface chemistry that has been successfully adapted to analyze mineralized tissue
such as dentin (22). It consists of ion gun pulsing high-energy ions into the target
surface. This bombardment causes collision cascades, disrupting atoms in the top
1 or 2 monolayers, causing an ejection and emissions of secondary ions from the surface. The charged ion mass is determined by the flight time from the surface to the
detector (23). Mass spectra with multiple peaks that arise as a result of molecular

From the *Discipline of Endodontics, Faculty of Dentistry, University of Toronto; and Department of Chemical Engineering and Applied Chemistry, University of
Toronto, Toronto, Canada.
Address requests for reprints to Dr Bettina R. Basrani, Co-Director MSc Endodontic Program, Faculty of Dentistry, University of Toronto, 124 Edward Street, Room
348C, Toronto, ON, M5G 1G6 Canada. E-mail address: Bettina.basrani@dentistry.utoronto.ca
0099-2399/$ - see front matter
Copyright 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2014.08.017

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fragmentations can then be analyzed or compared with SIMS library to
obtain parent molecular information. When subjected to TOF-SIMS
analysis, NaOCl/CHX precipitate yields peaks at 127, 153, 170, and
195 u, PCA yields a specific 127-u peak, and CHX yields a parent molecule peak at 505 u (18).
The penetration of the precipitate and PCA into DTs has not been
determined. If PCA is formed inside DTs, this may act as a reservoir of
PCA even if attempts are made to eliminate its formation in the main root
canal. Therefore, the aim of this study was to determine the formation of
precipitate and PCA on the surface and in the tubules of dentin irrigated
with NaOCl and subsequent irrigation with EDTA, NaOCl and CHX, or
saline and QMiX by using qualitative TOF-SIMS analysis.

Materials and Methods

re-embedded in low viscosity resin with the DT now oriented parallel

to the surface. The resin was again shaved from the surface to expose
DT in length to determine presence and depth of precipitate. The surfaces of the longitudinal sections prepared for each group (group 1,
CHX and group 2, QMiX) were again analyzed with TOF-SIMS in BA
imaging mode by using the previously stated conditions.

Results
Precipitate Formation
A brown precipitate was visually observed on the cross-sectional
surfaces of all group 1 samples immediately after CHX immersion. No
color change or precipitate was visualized on the cross-sectional surface of group 2 samples after immersion in QMiX.

Preparation of Samples
The study was approved by the universitys Health Sciences
Research Ethics Board. Two-millimeter-thick horizontal slices of
mid-root dentin were obtained from 4 extracted non-carious human
upper molars that had been previously stored in methyl alcohol by using
a diamond-coated saw (Leica EM TXP Target Sectioning System; Leica
Microsystems GmbH, Vienna, Austria). The slices were split vertically
to create 12 dentin blocks. Blocks were embedded in low viscosity
epoxy resin (Epo-Thin; Buehler, Lake Bluff, IL) and allowed to set
for 24 hours. The resin covering the root canal aspect of the embedded
block was then carefully removed with a diamond blade (Leica EM UC6/
FC6 Ultra-cryomicrotome; Leica Microsystems GmbH) to expose dentin,
with the DTs oriented approximately perpendicular to the exposed
surface.

TOF-SIMS Analysis: Cross Sections

In positive ion mass spectra analysis, intense peaks for residual
CHX and CHX breakdown products, including PCA, were detected on
the surface of all group 1 (CHX) blocks (Fig. 1A). In negative ion
mass spectra analysis, intense peaks of chlorine (Cl ), but not PO2
and PO3 , were detected on the surface (Fig. 1B). An irregular precipitate occluding nearly all DTs could be seen on the surface in BA mode
(Fig. 2A and B).
In positive ion mass spectra analysis, no precipitate, CHX, or CHX
breakdown products, including PCA, were detected on the dentin surface of any of the group 2 (QMiX) blocks (Fig. 1C). In negative ion mass
spectra analysis, significant peaks were seen for PO2 , PO3 , and Cl
(Fig. 1D). Patent DT with an absence of precipitate could be seen on the
surface in BA mode (Fig. 2C and D).

Treatment of Samples
Blocks were randomly divided into 2 groups. For group 1 (CHX),
6 blocks were immersed in 5 mL 2.5% NaOCl (sodium hypochlorite;
Lavo Inc, Montreal, QC, Canada) for 3 minutes, followed immediately
by 5 mL 17% EDTA (Vista Dental Products, Racine, WI) for 1 minute,
fresh 5 mL 2.5% NaOCl for 2 minutes, and a final immersion in 5 mL
2% CHX (chlorhexidine digluconate BP; Medisca, Montreal, QC, Canada) for 1 minute. For group 2 (QMiX), 6 blocks were immersed in
5 mL 2.5% NaOCl for 3 minutes, followed immediately by immersion
in 5 mL sterile saline (Aqualite; Hospira, Montreal, QC, Canada) (per
manufacturers recommendation) and then 5 mL QMiX (Dentsply Tulsa
Dental, Tulsa, OK) for 1 minute. Samples were dried on a bench top
overnight.

TOF-SIMS Analysis: Longitudinal Sections

In positive BA imaging, breakdown products of CHX and a consistent reading for PCA were visualized on the surface and in the DTs of all
group 1 (CHX) blocks (Fig. 3A). In negative BA imaging, Cl was present on the surface and in the DTs of all group 1 (CHX) blocks (Fig. 3B).
In positive BA imaging, no breakdown products of CHX, including
PCA, were visualized on the surface or in the DTs of any of the group 2
(QMiX) blocks (Fig. 3C). Under negative BA imaging, Cl could not be
visualized on the surface or in the DTs of any of the group 2 (QMiX)
blocks (Fig. 3D).

TOF-SIMS Analysis: Cross Sections

The surface analysis of 3 blocks from each group was performed
by TOF-SIMS (TOF-SIMS IV; ION-TOF GmbH, Munster, Germany). A bismuth (Bi) liquid metal ion gun was used as the primary ion source by
using the Bi3++ cluster. The gun was operated in a high-mass-resolution
bunched mode over an area of 500  500 mm for 100 seconds. In addition, spectra were obtained in a high-spatial-resolution imaging mode
(burst-alignment [BA]). Images (256  256 pixels) were obtained
from 20 scans over an area of 150  150 mm (23). Charge neutralization was achieved by using pulsed electron flood gun. Both positive
and negative polarity spectra were obtained. Mass-scale calibration
was obtained by clearly identifiable and well-spaced peaks for both
the positive and negative spectra.
TOF-SIMS Analysis: Longitudinal Sections
The remaining 3 blocks from each treatment group were removed from the resin with the diamond-coated saw, turned 90o, and
JOE Volume 40, Number 12, December 2014

Discussion
Up until the time this article was written, there had been no published studies of TOF-SIMS analysis of human dentin subjected to new
irrigation modalities. In addition, no published articles explored the
presence or absence of PCA inside DTs or on dentin surface without first
removing the precipitate for testing (24, 25).
QMiX is a novel root canal irrigant formulated to remove
smear layer and enhance root canal disinfection (12, 13). It is
recommended that it be used as the final irrigant after irrigation with
NaOCl and saline (26). Its composition is proprietary, but the manufacturer has disclosed that it contains EDTA, CHX, and a surface-active
detergent (12). In view of the previous reports highlighting the interaction between CHX and NaOCl (1820, 24, 25, 27) and the formation of
PCA with its known toxicity (2830), this study set out to examine
whether a potential for such precipitation existed when QMiX
inadvertently came in contact with NaOCl during clinical use.
TOF-SIMS is mostly used as a qualitative study tool with fragmentation pattern peaks or fingerprinting analysis. TOF-SIMS technique
was chosen for this study because of the advantages of great surface
sensitivity, which can measure composition of 12 monolayers. It

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Figure 1. Selected TOF-SIMS mass spectra of dentin surfaces with high mass resolution: (A) positive ion of group 1 (CHX), (B) negative ion of group 1 (CHX), (C)
positive ion of group 2 (QMiX), and (D) negative ion of group 2 (QMiX). Marked are the positions of peaks assignable to CHX+ at 505 u (right inset); breakdown
products of CHX at 153, 170, and 195 u, including PCA+ at 127 u, Cl at 35 and 37 u, PO2 at 63 u, and PO3 at 79 u. Note relative abundance of PCA+ in group 1,
absence of PO2 and PO3 in group 1, absence of PCA+ in group 2, and abundance of Cl in groups 1 and 2.

exhibits good spatial and mass resolutions and provides chemical mapping of the samples with analysis of the images. However, TOF-SIMS
is sensitive to contamination and demonstrates difficulty in data
quantification. This latter disadvantage implies that the intensities of
individual peaks of the ions cannot be compared among different
samples (23, 27).

It may be argued that addition of a saline irrigation after NaOCl and

before CHX prevents formation of the precipitate. However, it has been
shown that the use of saline could reduce but not eliminate the formation of PCA in a root canal system (24, 25). Therefore, group 1 (CHX)
was chosen as a positive control group, where PCA was expected to be
produced. Also, EDTA irrigation was added in this group to ensure that

Figure 2. High-spatial-resolution (BA) TOF-SIMS images of ion distribution on dentin surface: (A) positive ion of group 1 (CHX), (B) negative ion of group 1
(CHX), (C) positive ion of group 2 (QMiX), and (D) negative ion of group 2 (QMiX). Total shows raw image; ClC6H4NH2+ shows distribution of PCA. Note
irregular precipitate, containing PCA, on surface of group 1 and smooth appearance with patent DTs in group 2. mc, maximum ion count in 1 pixel.

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Figure 3. High-spatial-resolution (BA) TOF-SIMS images of ion distribution in longitudinal sections of dentin: (A) positive ion of group 1 (CHX), (B) negative ion
of group 1 (CHX), (C) positive ion of group 2 (QMiX), and (D) negative ion of group 2 (QMiX). Pulp space is on topmost and dentin bottom-most in each image.
Total shows raw image; ClC6H4 H2N+ + ClC6H4CH2N2+ show distribution of PCA and CHX breakdown products, and Cl + 37Cl show distribution of chlorine.
Note irregular precipitate on surface (green arrows), the extension of PCA and CHX breakdown products, in addition to chlorine, into DTs in G1 (yellow arrows),
and lack thereof in group 2. mc, maximum ion count in 1 pixel.

the tubules remain open because microtomed dentinal blocks could

have a smear layer present as manufacturing side effect. In group 2
(QMiX), EDTA irrigation was not used because EDTA is supposedly
included in QMiX (12).
In dentin blocks treated in accordance with the protocol used in
group 1 (CHX), a precipitate formed (1820, 24, 27, 3133) and was
present on the dentin surface as had been described in previous
publications (21, 24). The precipitate occluded the DT (21) to the
extent that it prevented detection of dentin as evidenced by lack of emission of PO2 and PO3 ions from the surface. This precipitate was
shown to contain PCA in some studies (1820, 24, 25, 27) but not
in others (3133). It could be argued that the inability to identify
PCA was related to the use of different detection techniques, with a
reduced sensitivity to low-molecular-weight products or the use of solvents that may have undermined PCA detection (3133).
TOF-SIMS analysis of group 1 (CHX) blocks yielded a 127-u peak
characteristic of PCA. An analysis of CHX alone yields intense peaks at
127, 153, 170, and 195 u in addition to 505 u, its parent molecule.
This fingerprint of CHX and the relative intensities of its different
peaks are altered when CHX is broken down (18, 27). The
precipitate formed on dentin when NaOCl/CHX were mixed showed a
low parent peak and a more intense peak assignable to PCA. This
highlights the previously identified degradation of CHX exposed to
NaOCl and the formation of its degradation products (18, 27). In
group 2 (QMiX) no precipitate could be detected on the dentin
surface, indicating its absence or its presence below detectable levels.
The absence of a PCA containing precipitate could be considered a
positive finding when QMiX is used, considering its toxic potential
(2830). It also represents a confirmation of its protocol for use,
which advises a saline rinse after NaOCl and before QMiX (26).
JOE Volume 40, Number 12, December 2014

We found the presence of PCA and Cl containing products, which

would include CHX and PCA, in the DTs of blocks exposed to CHX after
NaOCl (group 1). This finding was not unexpected because both NaOCl
and CHX have the ability to enter DTs, as has been shown in previous
studies that have used dyes (14, 15) or measured their antibacterial
penetration (16, 17, 34). In this study we were not able to detect a
peak characteristic of CHX or its breakdown products inside the DTs
of all blocks in the QMiX (group 2) group. This was surprising
because previous studies have shown that it was effective in destroying
bacteria in DTs 500 mm below the surface (16, 34). We also found a
consistent absence of Cl in the DTs of the QMiX group. This could
reflect a low concentration of CHX in QMiX or possibly that a product
consistent with detergent, as reflected by the presence of peaks at 128,
142, 156, 170, 184, and 198 u, on the dentin surface (Fig. 1C) blocked
detection of CHX or inhibited its penetration into the DTs.
With TOF-SIMS analysis, it needs to be stressed that artifacts can
occur because of the manner in which the dentin blocks were handled.
Regardless of this possibility, however, the finding of a PCA containing
precipitate in all group 1 (CHX) blocks and none in all group 2 (QMiX)
blocks left little doubt that the findings were correct.
Finally, even though there is still debate whether a precipitate
containing PCA is present in the root canal after it is irrigated with
CHX and NaOCl, its potential as a carcinogen in animal models, its cytotoxicity on human cells, and its ability to cause methemoglobinemia
in neonates (2830) make it imperative that every precaution be
exercised to avoid its production. It is for that reason that we
endorse the manufacturers recommendation that a saline rinse be
used in the root canal after NaOCl and before the final rinse with
QMiX (26), or that distilled water and ethanol be used as an intermediate alternative (24, 25).

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Conclusion
Within the limitations of this study, PCA containing precipitate was
formed on the surface and in the tubules of dentin irrigated with NaOCl
followed by CHX. No precipitate or PCA was detected in the tubules of
dentin irrigated with NaOCl followed by saline and QMiX.

Acknowledgments
The authors thank Surface Interface Ontario for its assistance
in the conduct of these experiments and also Drs Torneck and
Friedman for their feedback on the manuscript.
This study was supported by a grant from Canadian Academy
of Endodontics Endowment Fund and Endo Tech.
The authors deny any conflicts of interest related to this study.

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