Forced degradation of L-(+)-bornesitol, a bioactive marker of Hancornia speciosa: Development and validation of stability indicating UHPLC-MS method and effect of degraded products on ACE inhibition

Article abstract of DOI:10.1016/j.jchromb.2018.06.045

The antihypertensive activity of the medicinal plant Hancornia speciosa has been previously demonstrated by us, being the activity ascribed to polyphenols and cyclitols like L-(+)-bornesitol. We herein evaluated the stability of the bioactive marker bornesitol submitted to forced degradation conditions. Bornesitol employed in the study was isolated from H. speciosa leaves. An UHPLC-ESI-MS/MS method was developed to investigate bornesitol stability based on MRM (Multiple Reaction Monitoring) acquisition mode and negative ionization mode, employing both specific (m/z 193 → 161 Da) and confirmatory (m/z 193 → 175 Da) transitions. A gradient elution of 0.1% formic acid in water and acetonitrile was performed on a HILIC column. The method was validated and showed adequate linearity (r² > 0.99), selectivity, specificity, accuracy, and precision (RSD < 2.9%). The method was robust for deliberate variations on dessolvation temperature, but not for changes in the flow rate and dessolvation gas. The results from the stability studies allowed us to classify bornesitol as labile for acidic and alkaline hydrolysis, but as very stable for oxidative and neutral hydrolysis exposure. Bornesitol was categorized as practically stable under photolysis degradation, whereas a considerable reduction on its contents was induced by metal ions and thermolysis exposure. Degraded samples from neutral hydrolysis and thermolysis were assayed in vitro for ACE inhibition and showed a substantial decrease in biological activity as compared to intact bornesitol. myo-Inositol was identified as the major degradation products in both matrices. This is the first report on bornesitol stability under different stress conditions and the obtained data are relevant for the development and quality control of standardized products from H. speciosa leaves.

Full text of DOI:10.1016/j.jchromb.2018.06.045

JournalofChromatographyB1093–1094(2018)31–38
Contents lists available at ScienceDirect
Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb
Forced degradation of L-(+)-bornesitol, a bioactive marker of Hancornia
speciosa: Development and validation of stability indicating UHPLC-MS
method and effect of degraded products on ACE inhibition
José Hugo de Sousa Gomesa, Grazielle Caroline da Silvab, Steyner F. Côrtesc,
Rodrigo Maia de Páduaa, Fernão Castro Bragaa,⁎
a
Departament of Pharmaceutical Products, Faculty of Pharmacy, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, Brazil
Departament of Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, Brazil
Departament of Pharmacology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, Brazil
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
The antihypertensive activity of the medicinal plant Hancornia speciosa has been previously demonstrated by us,
being the activity ascribed to polyphenols and cyclitols like L-(+)-bornesitol. We herein evaluated the stability of
the bioactive marker bornesitol submitted to forced degradation conditions. Bornesitol employed in the study
was isolated from H. speciosa leaves. An UHPLC-ESI-MS/MS method was developed to investigate bornesitol
stability based on MRM (Multiple Reaction Monitoring) acquisition mode and negative ionization mode, em-
ploying both specific (m/z 193 → 161 Da) and confirmatory (m/z 193 → 175 Da) transitions. A gradient elution
of 0.1% formic acid in water and acetonitrile was performed on a HILIC column. The method was validated and
showed adequate linearity (r2 > 0.99), selectivity, specificity, accuracy, and precision (RSD < 2.9%). The
method was robust for deliberate variations on dessolvation temperature, but not for changes in the flow rate
and dessolvation gas. The results from the stability studies allowed us to classify bornesitol as labile for acidic
and alkaline hydrolysis, but as very stable for oxidative and neutral hydrolysis exposure. Bornesitol was cate-
gorized as practically stable under photolysis degradation, whereas a considerable reduction on its contents was
induced by metal ions and thermolysis exposure. Degraded samples from neutral hydrolysis and thermolysis
were assayed in vitro for ACE inhibition and showed a substantial decrease in biological activity as compared to
intact bornesitol. myo-Inositol was identified as the major degradation products in both matrices. This is the first
report on bornesitol stability under different stress conditions and the obtained data are relevant for the de-
velopment and quality control of standardized products from H. speciosa leaves.
L-(+)-Bornesitol
Forced degradation
LC-MS analysis
MRM
ACE inhibition
myo-Inositol
1. Introduction
NF-κB inhibitors of H. speciosa, potentially useful as chemopreventive
agents [8]. The anti-diabetic potential of the species was recently re-
Hancornia speciosa Gomes (Apocynaceae) is a medicinal plant vul-
garly named “mangaba” in Brazil, traditionally used to treat diabetes
and hypertension, among other diseases [1]. We have previously in-
vestigated some of the medicinal properties ascribed to the species. The
potential anti-hypertensive activity of the leaf extract was demon-
strated by in vitro inhibition of angiotensin I-converting enzyme (ACE)
[2] and vasodilator effect on rat aortic [3] and mesenteric preparations
[4]. L-(+)-Bornesitol was identified as the main ACE-inhibiting con-
stituent of H. speciosa [5]. The hypotensive effect of a standardized
fraction derived from the extract, enriched in L-(+)-bornesitol and
rutin, was further demonstrated in normotensive [6] and hypertensive
mice [7]. Moreover, bornesitol, quinic acid, and rutin were identified as
ported by promoting an increase in glucose uptake and alpha-glucosi-
dase inhibition [9]. The cyclitol L-(+)-bornesitol plays a key role in the
anti-hypertensive effect elicited by H. speciosa and is therefore con-
sidered its bioactive marker.
L-(+)-Bornesitol (1) is a methylated derivative of myo-inositol, a
compound found in different plant species [10]. Its physiological and
biological functions are related to the storage of seed products, stress-
related responses, production of inositol-glycosides, and osmor-
egulatory activity [11].
⁎
Corresponding author.
E-mail address: fernaobraga@farmacia.ufmg.br (F.C. Braga).
https://doi.org/10.1016/j.jchromb.2018.06.045
Received 27 March 2018; Received in revised form 10 May 2018; Accepted 20 June 2018
Availableonline22June2018
1570-0232/©2018PublishedbyElsevierB.V.

J.H.d.S. Gomes et al.
JournalofChromatographyB1093–1094(2018)31–38
Aldrich (St. Louis, Missouri, USA). HPLC grade solvents (methanol and
acetonitrile) were obtained from Tedia (Fairfield, Ohio, USA) and
deionized water was produced by
a Milli-Q system (Millipore,
Burlington, Massachusetts, USA). 30% (v/v) hydrogen peroxide solu-
tion was obtained from Synth (Diadema, Brazil), whereas potassium
permanganate, cooper sulfate, chloridric acid, ethyl acetate, acetic acid,
formic acid, sulfuric acid, silica gel 60 G, silica gel 60,230–400 mesh,
and silica gel 60 70–230 mesh were purchased from Merck (Darmstadt,
Germany). Activated charcoal was furnished by Synth (Diadema,
Brazil) and sodium hydroxide by Fmaia (Cotia, Brazil). Solvents of
analytical grade isopropyl alcohol, n-butyl alcohol, acetone, methanol,
and ethanol 95 °GL were obtained from Fmaia (Cotia, Brazil). Ethyl
alcohol 92 °GL was purchased from Emfal (Betim, Brazil).
(1)
The World Health Organization rates hypertension as one of the
most important causes of premature death worldwide and 1.56 billion
adults are estimated to be living with high blood pressure in 2025 [12].
Hypertension management involves both non-pharmacological and
pharmacological approaches [13] and numerous drugs are currently
available to treat this condition. However, low adherence to treatment
mainly results from the appearance of side effects, as well as to the
inadequate restoring of blood pressure, thus demanding the develop-
ment of new antihypertensive agents [13]. H. speciosa may be regarded
as a promising species for the development of an anti-hypertensive
phytomedicine, whereas its bioactive marker L-(+)-bornesitol may
constitute a valid chemical lead for drug development in view of its
pharmacological effects demonstrated so far.
Stability is a key quality requirement for a pharmaceutical product;
therefore, chemical stability assessment is a fundamental step during
hit selection for preclinical development [14]. Likewise, stability stu-
dies of bioactive markers are also mandatory in preclinical studies,
before the product enter phase I clinical trials. A literature survey in-
dicates few stability studies carried out with natural products and, as
far as we know, bornesitol is not part of the list. Therefore, we herein
investigate the chemical stability of L-(+)-bornesitol in a forced de-
gradation study, employing different stress conditions. The results here
reported will enable predicting the conditions that must be strictly
controlled during bornesitol handling in order to guarantee long shelf
life to the finished products containing this cyclitol.
Some chromatographic methods have been proposed for bornesitol
quantitation, including those based on GC-FID [15], GC–MS [16], LC-
DAD with previous derivatization [1], and LC-RI [17], but they all
present limitations to be used in degradation studies. The quantitation
of bornesitol and other cyclitols is not straightforward in view of the
lack of proper chromophore groups, impairing the use of UV detection.
Therefore, we also developed and validated a HILIC-based UHPLC-ESI-
MS/MS method to quantify bornesitol accurately, with no interferences
from the degradation products. The developed method can be adapted
to quantify bornesitol in different matrices, including plasma, and used
in pharmacokinetic studies and clinical trials. The method can be also
adapted to the analysis of other structure-related cyclitols and sugars in
plant extracts. Finally, we evaluated the influence of bornesitol de-
gradation on ACE-inhibiting activity.
2.3. Bornesitol isolation
A portion (5.0 g) of the crude extract (Section 2.1) was suspended in
50.0 mL MeOH and 400.0 mL of EtOAc were added; this solution was
kept at −4 °C for 24 h. Afterwards, the suspension was centrifuged
(822 ×g; 20 min). The precipitate was collected, the supernatant was
dried to residue in a rotatory evaporator and was re-extracted using the
same procedure three times. In the sequence, the precipitate (3.5 g) was
dissolved in 35 mL of 0.1% phosphoric acid cooled at 4 °C, activated
charcoal (350 mg) was added to the solution, and shaken in a vortex for
5 min. The preparation was centrifuged at 4 °C (822 ×g, 20 min), the
supernatant was recovered and submitted to partitioning with ethyl
acetate-n-butanol-isopropanol (7:1:2, v/v) (2 × 100 mL) to afford the
aqueous and organic phases. Following, the aqueous phase was lyo-
philized and a portion (1 g) of it was subjected to filtration over silica
gel (0.04–0.063 mm), using a gradient elution of EtOAc, EtOAc/MeOH
and MeOH. According to TLC analysis (mobile phase: H2O/EtOAc/i-
PrOH (6:11:83, v/v); stationary phase: silica gel; spray reagent: KMnO4
0.5% w/v in NaOH 1.0 N), bornesitol concentrated in fractions eluted
with EtOAc/MeOH (85:15 and 80:20, v/v). The fractions were com-
bined, the solvent was removed, and the resulting solid was submitted
to crystallization from MeOH to furnish bornesitol as white needles
(66.3 mg). Its purity was checked by TLC, UHPLC-ESI-MS, UHPLC-DAD,
1H and 13C NMR analyses, as well as by melting point data, and was
concluded to be higher than 98%.
2.4. Instrumentation and chromatographic conditions
2.4.1. LC-MS/MS analysis
Analyses were performed on a Waters ACQUITY UPLC system
(Waters, Milford, Massachusetts, USA), composed of binary pump, auto
sampler, in-line degasser and photodiode array detector (Waters,
Milford, Massachusetts, USA). The data were processed using
MassLynx4.1 (Waters, Milford, Massachusetts, USA). Separation was
performed on an Acquity UPLC BEH HILIC (150 × 2.1 mm i.d.; 1.7 μm;
Waters, Milford, Massachusetts, USA) in combination with an Acquity
UPLC BEH HILIC guard column (5 × 2.1 mm; 1.7 μm; Waters, Milford,
Massachusetts, USA). The mobile phase consisted of 0.1% formic acid in
water (A) and acetonitrile (B), used in the following elution/re-equili-
bration conditions: 0–5.25 min, 13% B; 5.25–5.50 min, 75% B;
5.50–9.00 min; 75% B; 9.00–9.25, 13% B; 9.25–15.00 13% B, at a flow
rate of 0.3 mL/min, at 20 °C. The injection volume varied from 1.0 to
3.0 μL.
2. Material and methods
2.1. Plant material and extract preparation
H. speciosa leaves were collected in November 2014 in the munici-
pality of Montes Claros (geographic coordinates: 16°47′15″ S and
43°53′34″ W, altitude of 669 m). The plant species was identified by Dr.
Maria das Dores Magalhães Veloso and a voucher specimen is deposited
at Herbarium Montes Claros (UNIMONTES), under number 3816. The
leaves were dried in a ventilated oven at 45 °C for 48 h and ground in a
knife mill. The extracts were prepared by percolation of 100 g of the
dried material with ethyl acetate-methanol (1:1, v/v). The solvent was
removed by evaporation in a rotatory evaporator at maximal tem-
perature of 50 °C, resulting in a dark green extract (40.3 g).
2.4.2. Instrumentation conditions
A mass spectrometer Xeco™ Triple Quadrupole MS (Waters Corp.,
Milford, Massachusetts, USA) equipped with an electrospray ionization
(ESI) source, operating in negative ionization mode, was used in the
analysis. The capillary voltage was set to 3500 V, cone gas voltage 27 V,
and source temperature 120 °C. The cone gas flow was set to 90 L/h and
desolvation gas flow to 900 L/h at 450 °C. The data were accomplished
in the multiple reaction mode (MRM), using both specific (collision
2.2. Reagents and chemicals
Anhydrous pyridine and myo-inositol were purchased from Sigma-
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J.H.d.S. Gomes et al.
JournalofChromatographyB1093–1094(2018)31–38
energy 14 V) and nonspecific (collision energy 10 V) transitions.
was extracted (Section 2.5; samples from acid, alkaline, and ionic
medium degradation) or dried to residue in a centrifugal vacuum
concentrator at room temperature for 24 h (all other samples) and then
dissolved to 5 mL MeOH for LC-MS analysis.
2.5. Sample extraction
Matrix effect was observed in samples submitted to forced de-
gradation in acid, alkaline, and ionic medium. Such samples were
submitted to an extraction procedure to avoid matrix interference. The
samples were freeze-dried (−52 °C, 400 mm Hg, 48 h), the resulting
residue was dissolved in 1.5 mL anhydrous pyridine, and the solution
was sequentially submitted to sonication in an ultrasound bath
(20 min), vortex agitation (5 min), and centrifugation (6708 ×g,
10 min). The supernatant (1.0 mL) was collected and dried to residue in
2.7.1. Acidic and alkaline hydrolysis
The acidic and basic hydrolyses were evaluated in three grades. In
the mild condition, bornesitol was dissolved in HCl (1.0 mL; 0.1 M) or
NaOH (1.0 mL; 0.1 M) and the solution was kept under reflux for 8 h,
whereas in the medium condition the reflux time was extended for 24 h.
In the harsh condition, bornesitol was refluxed in HCl (1.0 mL; 5.0 M)
or NaOH (1.0 mL; 5.0 M) for 24 h. Following, the hydrolysis medium
was neutralized with NaOH (0.1 M) or HCl (0.1 M).
a
centrifugal vacuum concentrator Labconco A450 (Kansas City,
Missouri, USA) at room temperature for 24 h. The residue was diluted
to 5.0 mL MeOH, filtered over a PVDF membrane (0.22 μm), and in-
jected onto the UHPLC system.
2.7.2. Neutral hydrolysis and oxidative stress
The experiments were undertaken in mild, medium and harsh
conditions, without agitation. For neutral hydrolysis, 1.0 mL of an
aqueous solution of bornesitol was refluxed for 1, 2 and 5 days, re-
spectively. For oxidative stress, 0.5 mg of the drug was exposed to
1.0 mL of 3%, 10%, and 30% H2O2 (v/v) for 6, 24, and 48 h at room
temperature (R.T.), respectively.
2.6. Method validation
The method was validated according to AOAC [18] and European
Commission Decision [19] guidelines.
2.6.1. Specificity
2.7.3. Photolysis and thermolysis
To assess specificity, different samples were analyzed using the es-
tablished MRM conditions: blank (MeOH), matrices of degradation
conditions (acid, alkaline, peroxide and metal ions), and bornesitol
spiked with structurally related compounds (glucose and myo-inositol;
1.0 mg/mL) or with constituents found in H. speciosa leaves (chloro-
genic acid, rutin, caffeic acid and quercetin; 1.0 mg/mL) (n = 6) [19].
Solid samples of bornesitol were employed in both conditions.
Photolytic degradation was evaluated in two grades: 1.2 × 106 and
6.0 × 106 lx h. For thermolytic stress, bornesitol samples were kept at
70 °C for 5 days under light-shielding conditions in non-ventilated oven.
2.7.4. Metal ions
Bornesitol was dissolved in 1.0 mL of 0.05 M CuSO4 aqueous solu-
tion and it was kept under light-shielding conditions for 24 days.
2.6.2. Linearity, precision, limits of quantification (LOQ) and detection
(LOD)
A methanol solution of bornesitol (0.1 mg/mL) was used to build
calibration curves. A five-point calibration curve was obtained by the
injection, in triplicate, of different volumes (1.0 to 3.0 μL) of the stan-
dard solution and plotting the injected mass (100, 150, 200, 250, and
300 ng) against the peak areas on two separate days, using linear re-
gression (Prism 5; GraphPad Software, San Diego, California, USA).
The intraday precision was assessed by RSD values of sample solu-
tions analyzed on the same day, at the middle concentration point of
the calibration curve (n = 6). The inter-day precision was evaluated by
analyzing solutions prepared in two different days, by two different
analysts (n = 12), at same middle concentration. LOQ and LOD were
calculated according to ICH [20].
2.8. In vitro inhibition of angiotensin-converting enzyme (ACE)
Samples recovery from neutral hydrolysis and thermolysis experi-
ments had their ACE-inhibiting activity evaluated in vitro, using a pre-
viously described method [2]. Briefly, the assay was initiated by adding
10 μL of the samples (final concentration of 8.04 ng/mL [5]), 10 μL of
MeOH/HEPES (1:4) pH 8.15 (negative control), and 10 μL of captopril
solution (5 nM, positive control) into a 96-well plate (non-sterile, V-
botton). Rat serum (Wistar, male) was used as ACE source. The plate
was homogenized and pre-incubated at 37 °C for 20 min. The enzymatic
reaction was initiated by the addition of 50 μL HEPES buffer (pH 8.15)
and 30 μL Hip-Gly-Gly solution (100 mM). After homogenization, the
mixture was incubated for 45 min at 37 °C. The reaction was stopped by
the addition of 0.3 M sodium tungstate (50 μL) and 0.32 M sulfuric acid
(50 μL) solutions. The plate was homogenized and rested for 10 min.
Then, 50 μL of buffer HEPES and 3 μL of TNBS were added. The plate
was kept in dark at room temperature for 20 min. The absorbance was
measured using a microplate reader (Epoch, BioTek, Winooski, Ver-
mont, USA) at 415 nm. The absorbance was read against a blank so-
lution prepared in a similar manner, except by the addition of the so-
dium tungstate and sulfuric acid solutions before the rat serum. The
assays were performed in octuplicate and the results are expressed as
percentage of inhibition (% inhibition = [100 − (A1 × 100 / Ac)]),
where A1 is the absorbance of the solution in the presence of an in-
hibitor (Asample - Ablank); and Ac is the absorbance of the negative
control solution (Acontrol - Ablank) [19].
2.6.3. Accuracy
To assess accuracy, a 5.0 M NaCl methanol solution was spiked with
known amounts of bornesitol at three concentration levels within the
analytical curve. The analyte extraction was performed as described in
Section 2.5 and the solutions were injected in triplicate onto the LC-MS
system for recovery calculation.
2.6.4. Robustness
Sample solutions prepared in MeOH (n = 6) were analyzed em-
ploying both the established conditions and under deliberated variation
of desolvation temperature (440 and 460 °C), flow rate (0.240 and
0.360 mL/min) and desolvation gas flow (840 and 960 L/h) [21]. The
obtained concentrations were compared by ANOVA followed by Tu-
key's test (p < 0.05).
3. Results and discussion
2.7. Forced degradation conditions
Bornesitol is considered the bioactive marker of H. speciosa since it
contributes to the hypotensive effect elicited by the extract and fraction
from the leaves [2–7]. Stability studies are mandatory to new drugs and
to bioactive markers, before a product enter phase I clinical trials.
Stability is directly related to the safety and efficacy of a drug, once a
A 500 μg sample of bornesitol was employed in each degradation
condition. The experiments were carried out according to the Guidance
on Conduct of Stress Tests to Determine Inherent Stability of Drugs
[14]. After exposure to the degradation condition, the sample solution
33

J.H.d.S. Gomes et al.
JournalofChromatographyB1093–1094(2018)31–38
Fig. 1. Bornesitol deprotonated adduct and derived ions fragments observed in continuous infusion onto the MS spectrometer.
variety of degradation products can be formed under different condi-
tions, resulting from hydrolysis reactions across a wide range of pH,
humidity, temperature, oxidation and photolysis [14]. Therefore,
herein we investigated bornesitol stability to comprehend the condi-
tions that should be strictly controlled during its handling and storage,
as well as to evaluate the effect of degradation on the biological re-
sponse.
predicted deprotonated ion at m/z 193.1 Da [M-H]−. Two specific
fragment ions of bornesitol were observed using the established colli-
sion energy: the first at m/z 161 Da [M-H-CH3OH]− results from the
loss of methanol and the second at m/z 175 Da [M-H-H2O]− is formed
by dehydration (Fig. 1). All hydroxyl groups of bornesitol are located in
equatorial position, except the one neighbor to the methoxyl group,
which occupies an axial location, and is therefore more prone to be lost.
Although both ions were produced by continuous infusion of bor-
nesitol onto the MS equipment, only one specific fragment ion was
stable in the product ion spectrum of bornesitol: the one at m/z 161 Da.
Therefore, the MRM-MS method was developed to include the transi-
tion m/z 193 Da [M-H]− → m/z 161 Da [M-H-CH3OH]− (Fig. 2).
3.1. Bornesitol isolation
Bornesitol is not commercially available; therefore, firstly we had to
obtain it in a purity (> 98%) required for its use as a reference com-
pound. We have previously reported a chromatographic procedure to
isolate bornesitol from a crude ethanol extract of H. speciosa leaves [8].
Although it affords bornesitol at a satisfactory purity grade, the method
was shown to be time consuming and to employ large volumes of sol-
vents. Therefore, we developed an alternative and faster method for
bornesitol isolation, starting from an extract of H. speciosa leaves ob-
tained by percolation with EtOAc/MeOH (1:1, v/v). Percolation was
show to be a simple and fast method for extract preparation and also
provided a satisfactory yield (40% w/w after solvent evaporation). The
dry extract was dissolved in methanol, following the addition of ethyl
acetate to precipitate a solid enriched in bornesitol. In addition to the
cyclitol, the precipitate also contained phenolic compounds such as
rutin, according to chromatographic analysis. Activated charcoal was
employed to remove the phenolic interferents [22] and further pur-
ification was achieved by partitioning between immiscible solvents. An
additional purification was undertaken by filtration over silica gel, that
afforded some fractions enriched in bornesitol, which were combined
and recrystallized to give bornesitol (> 98% purity), with an overall
yield of 1.45%. Our previous method for bornesitol isolation [8], which
requires an initial fractionation of the crude extract by column chro-
matography over silica gel, afforded an overall yield of 0.46% (w/w).
As far as we know, there is no other report of bornesitol isolation from
H. speciosa described on literature. The purity of the isolated compound
was confirmed by TLC, melting point, 1H and 13C NMR data (Table S1,
Supplementary material).
3.3. Matrix effect and chromatographic conditions
MRM affords a considerable specificity to MS results. However, the
signal of bornesitol in the selected transition (m/z 193 → 161 Da) was
suppressed either in the presence of NaCl, produced after neutralization
of the medium in acid and alkaline hydrolysis, or CuSO4, employed in
metal ion degradation study, thus characterizing matrix effect (ME). ME
can be overcome by chromatographic, spectrometric, and sample pre-
paration approaches. We adopted the last tactic and evaluated different
solvents with high dielectric constant (ε) to extract bornesitol selec-
tively, and thus eliminate NaCl interference: N,N-dimethylformamide
(ε = 36.71), dimethylsulfoxide (ε = 46.7), and pyridine (ε = 12.4). The
last solvent promoted a selective recovery of bornesitol, removing NaCl
satisfactory. The suppression of bornesitol signal due to matrix effect
was not observed in all other forced degradation conditions evaluated
(data not shown).
3.4. Method validation
3.4.1. Specificity
The peak areas obtained for bornesitol in the analyzed matrices
were equivalent to the areas recorded for blank solutions spiked with
bornesitol. Additionally, the blank solutions did not show any inter-
ference in the MRM mode, suggesting specificity of the method for the
selected transition of bornesitol (data not shown).
3.2. LC-MS/MS method development
3.4.2. Linearity, precision, limits of quantification (LOQ) and detection
(LOD)
The chromatographic conditions were optimized aiming to elute
bornesitol and its degradation products in a short run. For this purpose,
the developed method comprised two steps of isocratic elution with
eluents of distinct strength. Firstly, elution was performed with 13%
ACN for 5.25 min to assure the elution of bornesitol and its degradants
of equivalent polarity. Secondly, the proportion of ACN was rapidly
increased to 75% and this proportion was kept until 9 min, to allow the
elution of low polar compounds that may have been formed.
The UHPLC-ESI-MS/MS determination of bornesitol was achieved
by operating the mass spectrometer in the MRM mode. The direct in-
fusion of bornesitol onto the MS equipment in the negative ion mode
exhibited a greater signal to noise ratio than the positive ion mode (data
not shown). Thereby, spectroscopic conditions were optimized for the
The calibration curves were obtained at two consecutive days and
were statistically equivalent (p > 0.05). The data were combined and
the resulting calibration curve was linear over the established range
(100–300 ng) with r2 of 0.9995 for the regression equation
(y = 2.18x + 14). Intra-day and inter-day precision showed RSD of
2.77% and 2.90%, respectively. The mean concentration of bornesitol
evaluated in the inter-day precision experiment did not differ statisti-
cally (p > 0.05). LOQ and LOD were respectively 28.14 ng and
9.39 ng.
3.4.3. Accuracy
The method is accurate with recovery rates from 93.83% to
34

J.H.d.S. Gomes et al.
JournalofChromatographyB1093–1094(2018)31–38
Fig. 2. MRM product ion spectrum in negative mode of ionization selected for bornesitol quantification. The MS/MS conditions were optimized to favor the transition
of the precursor ion at m/z 193.13 Da to the product ion at m/z 161 Da.
104.83% for NaCl sample spiked with bornesitol (Table 1). There is no
fixed value to consider recovery acceptable for quantitative analysis,
Table 1
Recovery rates of bornesitol spiked at three levels of concentration into 5.0 M
NaCl degraded matrices.
once it varies with the analyte concentration in the sample [18].
Level
1
Recovery (n = 3) (% w/w)
Mean (%)
99.12
RSD (%)
5.42
3.4.4. Robustness
Variations in the desolvation temperature did not affect bornesitol
quantitation (p > 0.05). On the other hand, different contents of bor-
nesitol were obtained for deliberate variations in the flow rate and
desolvation gas flow (p < 0.05). Hence, the method requires attention
in all operating conditions.
95.14
96.93
105.29
100.25
106.67
107.53
95.54
93.83
95.86
2
3
104.82
95.08
3.98
1.09
3.5. Chemical stability of bornesitol
3.5.1. Acidic and alkaline hydrolysis
Hydrolytic degradation is usually evaluated over a wide range of pH
to mimic situations that may occur in normal exposure during product's
shelf life [23]. In the present study, bornesitol stability was evaluated
under mild, medium, and harsh conditions, according to the time of
exposure and concentration of the stress agent and the results are
shown in Table 2.
Table 2
Contents of bornesitol in samples submitted to forced degradation, employing
different stress conditions.
Condition
Bornesitol (% w/w
Mild
s.d.)
Medium
Harsh
In degradation studies, a condition that results in 20–80% of drug
consumption should be achieved in order to classify the analyte ac-
cording to its stability [14]. This requirement was obtained in mild
conditions for acid and alkaline exposures, and bornesitol was classified
as labile or class III. The obtained data suggests that this cyclitol is
especially susceptible to acidic conditions (Table 2). As far as we know,
there are no previous reports on stability studies for bornesitol or re-
lated cyclitols like pinitol and myo-inositol. Moreover, the majority of
the studies reported for other classes of drugs are focused on the
identification of degradation products, and not on the extension of
decomposition. Nevertheless, some drugs have been submitted to si-
milar degradation conditions.
The stability of mesalamine was evaluated under acidic (2.0 M HCl;
3 h, room temperature-R.T.) and alkaline conditions (2.5 M NaOH, 3 h,
R.T.) and the extension of degradation was 67.75% and 64.52%, re-
spectively [24]. Complete degradation of tenofovir was observed under
acidic and alkaline exposures, immediately after the addition of the
stress agents at R.T. (HCl/NaOH) [25]. Chlorthalidone was evaluated
for acidic (0.1 M HCl, 60 min, 80 °C) and alkaline (0.1 M NaOH, 30 min,
Acidic
71.96
77.37
84.00
100.63
14.05
6.74
3.20
1.47
40.73
71.6
80.66
96.78
3.94
28.7
1.30
1.10
32.93
39.03
74.97
90.09
2.28
1.72
0.52
0.56
Alkaline
Neutral
Oxidative
80 °C) exposure, where 35.24% and 36.42% of decomposition were
found [26]. Olmesartan showed 60.15% and 27.54% of decomposition
in alkaline (0.01 NaOH, 5 min, R.T.) and acid conditions (1 M HCl, 2 h,
60 °C), respectively. In the same study, amlodipine decomposed 52.35%
in alkaline medium (1.0 M NaOH, 1 h, R.T.) and 28.5% in acidic con-
dition (1.0 M HCl, 2 h, 60 °C) [27]. Flubendazole displayed 99.25% of
decomposition when submitted to alkaline degradation (1.0 M NaOH,
5 h, 80 °C) and 33.10% in acidic hydrolysis (1.0 M, 80 °C, 5 h) [28]. All
described studies employed milder conditions than those here described
for bornesitol. Nevertheless, it showed high stability, which can be at-
tributed to the absence of common ionizable functional groups, such as
ethers, ketones, aldehydes, and amides [29]. It is important to stress
35

J.H.d.S. Gomes et al.
JournalofChromatographyB1093–1094(2018)31–38
that elevated decomposition rated was observed for bornesitol sub-
mitted to harsh stress conditions, which are very unlikely to be
achieved during its shelf-life.
40].
Photolysis can occur through oxidative or nonoxidative mechan-
isms. Oxidative photolysis demands singlet oxygen (1O2), which reacts
with unsaturated bonds, as found in alkenes, dienes, and polynuclear
aromatic hydrocarbons, or alternatively with the participation of triplet
oxygen (3O2), which reacts with free radicals of the substrate. Non-
oxidative photolysis involves dimerization, rearrangements, iso-
merization, decarboxylation, cyclization and hemolytic cleavage of X-C
hetero bonds [29]. Bornesitol would hardly undergo photolysis by an
oxidative mechanism due to the lack of the required functional groups
in the molecule, whereas non-oxidative mechanism apparently did not
occur in the experimental conditions.
3.5.2. Neutral hydrolysis and oxidative stress
The stability of bornesitol was evaluated over three grades (mild,
medium, and harsh), according to the time of exposure for neutral
hydrolysis, and based on the concentration of the oxidation agent and
time of exposure for the oxidative stress (Table 2).
The ideal exposure in neutral hydrolysis condition was achieved in
the harsh experiment. To be categorized as a class VI drug, none de-
gradation should be observed for the tested compound [14]; since 25%
of decomposition was detected, bornesitol was classified as class V or
stable drug over neutral water environments. As expected, a reduced
extension of decomposition was observed in neutral hydrolysis in
comparison to acidic and alkaline conditions. Most of the drugs in
clinical use are resistant to neutral degradation [25, 28, 30–34]. When
compared to drugs susceptible to undergo decomposition in neutral
medium [29, 35, 36], bornesitol was shown to keep its integrity in a
higher extent, probably due to absence of easily ionizable groups.
In oxidative stress, the extension of degradation required do classify
bornesitol according to stability was achieved under harsh exposure. It
was described as class V or stable compound over oxidative environ-
ments [14]. Bornesitol has been previously submitted to oxidation by
Acetobacter suboxydans [37]. According to the authors, the hydroxyl
group located at axial position is the preferential site for oxidation and
D-1-O-methyl-myo-inosose-2 was identified as the main degradation
product (2). The axial-hydroxyl group has been also identified as the
favored spot for oxidation in myo-inositol and its methyl esters deri-
vatives [38, 39]. Based on these previous findings, it is feasible to
suppose that oxidation of L-(+)-bornesitol occurs at the same point, i.e.
the axial-located hydroxyl group, leading to the previously described
monoketone derivative 2.
Thermolysis usually involves decarboxylation, isomerization, poly-
merization and rearrangements. The experiments are mostly under-
taken at 70 °C, a condition that furnishes important information about
drug stability [14]. Under this experimental condition, the content of
bornesitol was 79.70
1.13%. Most of the drugs in clinical use are
stable under thermal degradation, with decomposition rates below 10%
[25, 30–36, 40, 45–47]. It is important to emphasize that milder con-
ditions were employed in those studies than the condition applied to
bornesitol, which could explain the observed degradation rate.
3.5.4. Metal ions
Metal ions can catalyze several reactions and consequently improve
the magnitude of decomposition. Cu2+ can react with the analyte in
“outer sphere” (electron transfer) or “inner sphere” (ligand transfer)
[48] contributing to degradation. Bornesitol was exposed to a single
concentration of CuSO4, once variation in parameters such as tem-
perature and concentration is not considered relevant to this degrada-
tion agent [14]. After exposure, the content of bornesitol was
34.67
3.67%. Since the exposition was performed in aqueous
medium, it is feasible to suppose that Cu2+catalyzed the hydrolysis of
bornesitol.
3.6. ACE inhibition
Bornesitol and other structure-related cyclitols and sugars have
been shown to inhibit ACE [5], an enzyme that plays a key role in the
renin-angiotensin system, responsible for blood pressure regulation. In
order to evaluate the impact of forced degradation on ACE inhibiting
activity, bornesitol samples were submitted to neutral hydrolysis and to
thermolytic harsh exposure and the decomposed products had their
biological activity assayed in vitro. These two degradation conditions
were selected since they are more likely to occur during the shelf-life of
a drug.
(2)
Several drugs in clinical use have been shown to be more ex-
tensively degraded under milder oxidation conditions than bornesitol,
including mesalamine (54.84% degradation, 6% H2O2, 6 h, R.T.) [24],
tenofovir (immediate degradation, 0.3% H2O2, R.T.), emtricitabine
(12% degradation, 8% H2O2, 5 h, R.T.) [25], rilpivirine (7% degrada-
tion, 8% H2O2, 5 h, R.T.) [30], valsartan (62.6% degradation, 10%
H2O2, 2 h, R.T.) [40], and nebivolol hydrochloride (96.42% degrada-
tion, 3% H2O2, 8 h, R.T.) [41]. The stability of bornesitol over a wide
range of exposure to an oxidant agent could be rationalized from its
chemical structure. Oxidation involves electron transfer to generate
reactive anions and cations [23, 29]. Amines, benzylic carbon, allylic
carbon, tertiary carbon, sulphides, and phenols are susceptible groups
to undergo oxidation and they are absent in the cyclitol [42–44].
Moreover, the product from bornesitol oxidation has a sp2‑carbon atom
that enhances ring strain and makes it unstable. Therefore, further
oxidation of other hydroxyl groups is not favored.
3.5.3. Photolysis and thermolysis
Photolytic degradation was evaluated in two grades (1.2 and
6.0 million lx h), according to the intensity of light exposure at 354 nm
[14]. The content of bornesitol did not decrease substantially under
Fig. 3. In vitro ACE inhibition elicited by bornesitol (BOH; 41.4 μM) and its
decomposition products resultant from neutral hydrolysis (NH) and thermolytic
degradation (TD). Saline (S) was employed as blank and captopril (C) as posi-
tive control (5 nM). The results are reported as percentage of ACE inhibition
and are representative of eight experiments with ***p < 0.005 when com-
pared to bornesitol (BOH) (One-way ANOVA).
light exposure (respectively 99.43
0.87% and 98.78
1.5%) and it
was classified as practically stable, or class IV drug. Data from literature
indicates that most of the drugs are stable under photolytic conditions
[24–27, 32–34, 41, 45]; a few exceptions have been reported so far [30,
36

J.H.d.S. Gomes et al.
JournalofChromatographyB1093–1094(2018)31–38
Fig. 4. UHPLC-ESI-MS chromatograms registered in negative MRM mode of analysis in two channels for samples submitted to neutral hydrolysis and thermolysis
showing (A) bornesitol transition (m/z 193 → 161 Da), and (B) myo-inositol transition (m/z 179 → 86.9 Da). The chromatogram depicted in (C) corresponds to the
total ion count (bornesitol plus myo-inositol). Chromatographic and MS conditions: see Section 2.4.
In both evaluated conditions, bornesitol decomposition led to a
substantial decrease in ACE-inhibiting activity (Fig. 3). Although a
higher reduction in biological activity was observed under thermolysis,
the content of bornesitol quantified after thermal stress
validated to quantify bornesitol in degraded matrices. Method se-
lectivity was obtained by using specific and confirmatory MRM tran-
sitions. The method was applied to investigate, for the first time, the
stability of bornesitol over different stress conditions. Bornesitol was
shown to be labile for acidic and alkaline hydrolysis, but very stable for
oxidative and neutral hydrolysis exposure. It was categorized as prac-
tically stable under photolysis degradation, whereas a considerable
reduction on bornesitol contents was induced by metal ions and ther-
molysis exposure. The obtained data are relevant to support the de-
velopment and quality control of standardized anti-hypertensive pre-
parations from Hancornia speciosa leaves or bornesitol itself. Moreover,
we demonstrated for the first time the conversion of bornesitol into
myo-inositol after either thermolysis or neutral hydrolysis exposure and
the corresponding reduction in ACE-inhibitory activity.
(79.70
1.13%) did not vary substantially from the concentration
0.52%). In order to iden-
measured after neutral hydrolysis (74.97
tify the possible degradation products formed under both stress con-
ditions, the matrices were subjected to UPLC-ESI-MS/MS analyses ei-
ther in the positive and negative scan modes.
The total ion count (TIC) chromatogram registered for the ther-
molysis and neutral hydrolysis samples did not differ substantially (Fig.
S1, Supplementary material). Importantly, both chromatograms dis-
played a peak with retention time in the interval 2.0–3.5 min, ascribed
to the deprotonated ion at m/z 179.09 Da [M-H]− of myo-inositol (MW
180.16 g/mol) (Fig. S2, Supplementary material). In order to confirm
the attribution unambiguously, a MRM method was developed for myo-
inositol determination based on the transition m/z 179.09 Da → m/z
86.9 Da (collision energy 34 V) in ESI− (Fig. S3, Supplementary mate-
rial). The chromatograms were acquired in MRM mode employing two
channels to allow the simultaneous identification of bornesitol and myo-
inositol in the degraded samples (Fig. 4). The obtained chromatograms
exhibit co-eluted peaks of bornesitol and myo-inositol in the interval
2.0–3.5 min, confirming unequivocally the presence of myo-inositol in
the degraded samples. It is important to stress that those chromato-
grams were not used for quantification purpose, but just to confirm
myo-inositol formation.
Acknowledgements
We acknowledge the Brazilian agency CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico) (405219/2013-0) for the
financial support along with research fellowships (FCB, SFC). CAPES/
Brazil is acknowledged for a PhD fellowship (JHSG).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jchromb.2018.06.045.
Selectivity is not mandatory in analysis performed in MRM mode,
due to the high capability of the quadrupoles in alternating their
radiofrequency and selecting ions at a short period of time (in the range
of milliseconds). This feature allowed us to identity myo-inositol co-
eluted with bornesitol.
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