Stability and solubility of trans-resveratrol are strongly influenced by pH and temperature

Article abstract of DOI:10.1016/j.ejpb.2015.04.002

Recently trans-resveratrol (trans-RSV) has received great attention due to its prophylactic and therapeutic properties. Its limited bioavailability provides compelling evidence of the need for more suitable formulations in order to attain better clinical

Full text of DOI:10.1016/j.ejpb.2015.04.002

Accepted Manuscript
Stability and solubility of trans-resveratrol are strongly influenced by ph and
temperature
Špela Zupančič, Zoran Lavrič, Julijana Kristl
PII:
DOI:
Reference:
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http://dx.doi.org/10.1016/j.ejpb.2015.04.002
EJPB 11892
European Journal of Pharmaceutics and Biophar-
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Please cite this article as: Š. Zupančič, Z. Lavrič, J. Kristl, Stability and solubility of trans-resveratrol are strongly
influenced by ph and temperature, European Journal of Pharmaceutics and Biopharmaceutics (2015), doi: http://
dx.doi.org/10.1016/j.ejpb.2015.04.002
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STABILITY AND SOLUBILITY OF TRANS-RESVERATROL ARE
STRONGLY INFLUENCED BY pH AND TEMPERATURE
1
1
Špela Zupančič , Zoran Lavrič , Julijana Kristl1*
1 University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana, Slovenia
E-mails: spela.zupancic@ffa.uni-lj.si, zoran.lavric@ffa.uni-lj.si, julijana.kristl@ffa.uni-lj.si
* Corresponding author.
University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia.
Tel.: + 386 1 476 9521;
Fax: + 386 1 425 8031
E-mail: julijana.kristl@ffa.uni-lj.si
1

ABSTRACT
Recently trans-resveratrol (trans-RSV) has received great attention due to its prophylactic and
therapeutic properties. Its limited bioavailability provides compelling evidence of the need for
more suitable formulations in order to attain better clinical effectiveness. Some physicochemical
properties of trans-RSV are still unknown or research findings are contradictory. Therefore, this
paper presents newly determined trans-RSV solubility and stability at various pH and
temperatures, and the importance of such data for the studies of novel trans-RSV-loaded
nanofibers. In acidic pH trans-RSV was stable, whereas its degradation started to increase
exponentially above pH 6.8. Consequently, it is worthwhile to note that special consideration has
to be dedicated to long dissolution testing or biological assays on cell lines in order to obtain
relevant data. Measurements were done by validated UV/VIS spectroscopy, HPLC, and newly
developed UPLC methods. Specificity was confirmed for HPLC and UPLC method, whereas
UV/VIS spectroscopy resulted in false higher trans-RSV concentrations in conditions under
which it was not stable (alkaline pH, light, increased temperature). The study is of interest
because it draws attention to the importance of careful selected experimental conditions, their
influence on the trans-RSV stability and the implications this has for formulation development,
storage, and maintenance of therapeutic doses.
Keywords: resveratrol, stability, solubility, analytical methods, validation, isomerization,
degradation, nanofibers, release, drug delivery
Chemical compounds
Acetonitrile (PubChem CID: 6342); Chloroform (PubChem CID: 6212); Formic acid (PubChem
CID: 284); Methanol (PubChem CID: 887); Phosphoric acid (PubChem CID: 1004); Potassium
dihydrogen phosphate (PubChem CID: 516951); Sodium hydroxide (PubChem CID: 14798);
Sodium iodide (PubChem CID: 5238); Sodium tetraborate decahydrate (PubChem CID:
22435386); Trans-resveratrol (PubChem CID: 445154)
2

1 INTRODUCTION
Trans-resveratrol (3,5,4′-trihydroxystilbene; trans-RSV), one of the most promising naturally
occurring compounds, has been shown to exhibit a wide range of therapeutic and preventive
properties. Several studies have confirmed its anti-inflammatory, cardioprotective, antitumor,
neuroprotective, and antioxidant activities [1, 2]. Unfortunately, its in vivo applications are
severely limited due to low bioavailability as a consequence of its poor aqueous solubility,
limited stability, and extensive liver metabolism [2, 3]. The research community is working to
solve these problems by formulating new drug delivery systems that stabilize and protect trans-
RSV from degradation, increase its solubility in water, achieve sustained release, and target
trans-RSV to specific locations via multiparticulate and nanodelivery systems [4-7]. In addition,
trans-RSV has attracted attention not only from the pharmaceutical but also cosmetics [8] and
food [9] industries.
Although there are currently more than 6,830 publications referenced on the US National Library
of Medicine’s PubMed service about trans-RSV, some basic physicochemical properties of
trans-RSV are still unknown or the research findings frequently contradict each other.
Comprehensive studies of trans-RSV stability and solubility in water are scarce, but these
represent fundamental information required for developing a dosing regimen, preparing carrier
systems and their loading ability, and choosing experimental conditions for in vitro and in vivo
experiments. Some studies have claimed that trans-RSV has limited stability under the influence
of light, certain pH levels, and temperature, which can cause trans-RSV isomerization or
degradation [10]. Trans-RSV is highly photosensitive and rapidly isomerizes to cis-RSV when
exposed to ultraviolet or visible light [2]. Unfortunately, the less stable trans-RSV is more
biologically active [11]. Isomerization kinetics is affected by irradiation time, wavelength, the
physical state of trans-RSV, and temperature [12, 13].
By contrast, the effect of pH on trans-RSV stability has been investigated in very few studies.
Trela and Waterhouse in 1996 proved that trans-RSV is stable in buffers with pH 1, 3.5, and 7.0
for 28 days, whereas at pH 10 it rapidly degraded with a half-life of only 1.6 hours [12]. To the
best of our knowledge, there are no published data on trans-RSV degradation kinetics at specific
pH levels relevant to physiological fluids.
3

The impact of temperature on trans-RSV stability was studied at very different temperatures. For
example, Liazid et al discovered that when trans-RSV was exposed for 20 minutes to increased
temperatures the extent of degradation is 17% at 125 °C, 39% at 150 °C, and 70% at 175 °C [14].
On the other hand, Sessa et al. also evaluated trans-RSV stability and found a higher degradation
rate at 4 °C than at 30 and 55 °C during 30 days [9]. Comparing different studies’ findings shows
that trans-RSV in solution is not stable at either low or high temperatures [9].
Concerns have also been raised regarding the correct values of trans-RSV water solubility due to
different data observed in the literature. The data for trans-RSV solubility obtained by different
authors varies from 50 ng/L (at pH 6 and 8 at 37 °C) [15] to 19.6 mg/L (in water at 25 °C) [16] or
21 mg/L (at pH 7.4 and 37 °C) [17], despite the relatively similar experimental conditions.
For identifying and quantifying trans-RSV in solutions and plasma various analytical methods
have been used involving UV/VIS spectroscopy (UV/VIS) [13, 18, 19], high performance liquid
chromatography (HPLC) coupled with fluorescence, UV/VIS [9, 20-22] or mass spectrometry
[23] detection, and gas chromatography with mass selective detection (GC-MS) [24]. An
overview of the literature about trans-RSV showed that validations of the HPLC method were
usually performed for determining the trans-RSV content in wine [24] or in in vivo
pharmacokinetic studies [23], covering the samples with several metabolic or degradation
compounds. By contrast, in the reports that have dealt with formulation development, generally
no validation of analytical methods is specified, with a rare exception in which the validation was
provided and results reported [22].
Considering the low stability and solubility of trans-RSV, our hypothesis was that careful
selection of particular experimental conditions and analytical methods would improve the
consistency of findings. Therefore, a new analytical method, UPLC with photodiode array
detection (UPLC) for analysis of trans-RSV separated from cis-isomer and degradation products,
was developed and validated. The findings were compared with those of the UV/VIS and HPLC
methods commonly used. The trans-RSV stability and solubility were determined at the
conditions, which resemble as much as possible physiological or storage conditions. In addition,
as a case study, the release of trans-RSV from newly developed nanofibers was determined and
the influence of pH and the selection of analytical method on the release profiles was
investigated. All the findings are compared with some published data in order to determine the
4

key points for careful analysis, formulation, and preparation of products that consistently
maintain therapeutic dose of trans-RSV.
2 METHODS AND MATERIALS
2.1 Materials
Trans-RSV was purchased from LGC Standards GmbH, Germany with a declared purity of
99.6% according to the manufacturer. Orthophosphoric acid 85%, potassium dihydrogen
phosphate, formic acid, acetic acid, sodium chloride, sodium iodide, hydrochloric acid fuming
37%, sodium tetraborate decahydrate, sodium hydroxide, chloroform, and acetone (for analysis)
were obtained from Merck KGaA (Germany). Poly(ε-caprolactone) PCL (Mr 70.000–90.000
g/mol) was purchased from Sigma Aldrich (Germany). Acetonitrile and methanol of HPLC grade
were purchased from J. T. Baker (the Netherlands). Ultrapure water was obtained from a Milli-
Q® UF-Plus apparatus (Millipore Corp., Burlington, MA, USA) and used for preparing the
mobile phases. Deionized water (type 2, analytical grade) was obtained from Millipore Elix 35
Water Purification System (Millipore Corp., Burlington, MA, USA) and used throughout the
experiments. Buffers with pH 1.2, 6.8, and 7.4 were prepared according to Ph. Eur. Chapter
5.17.1. [25]. The pH of each freshly prepared buffer was adjusted to the prescribed pH using a pH
meter (220 SevenCompact™ pH/Ion meter, Mettler Toledo, Switzerland). Filters with pore size
0.2 µm (Minisart RC 15, Sartorius Stedim Biotech GmbH, Germany) were used prior to each
sample analysis. All RSV solutions were protected from light during the experiments unless
otherwise stated.
2.2 Standard solution preparation
Initial standard stock solutions of trans-RSV with concentrations of 1 mg/mL were further
diluted with water supplemented with 0.1% formic acid in order to obtain seven standard
solutions in the range of 0.5–15 µg/mL.
5

2.3 Analytical methods
2.3.1 UV/VIS spectroscopy
UV–VIS absorption spectra of the samples were recorded in the range of 240–370 nm with 1 nm
resolution using a UV/VIS spectrophotometer (Hewlett Packard 8453, Germany) at room
temperature. Maximal trans-RSV absorption in aqueous solution at 306 nm was used to calculate
the trans-RSV concentration.
2.3.2 HPLC method
Trans-RSV was analyzed using the HPLC method previously described by Bolko et al. with
small modifications [20]. Briefly, trans-RSV was determined with HPLC (Agilent 1100 Infinity
system, Agilent Technologies Inc., USA) using reverse phase column C18 (3.145 μm particle
size, 250x4.6 mm, YMC Europe GmbH, Germany) at 30 °C. The mobile phase was a mixture of
water and methanol 50:50 (v/v) supplemented with 0.25% (v/v) acetic acid. The auto-sampler
temperature was set at 4 °C. The injection volume was 20 μL, flow rate 1 mL/min, and run time
15 min. A UV scan was obtained from 240 to 370 nm with resolution 2 nm and area under the
curve of trans-RSV peak at 306 nm was chosen for detection and further calculation of the trans-
RSV concentration.
2.3.3 UPLC method
The UPLC method was developed to separate trans-RSV from cis-RSV and degradation products
with better resolution than with HPLC. Trans-RSV was determined by the chromatographic
system Acquity UPLC (Waters Corp., USA). A UV-VIS photodiode array module (PDA)
equipped with a high-sensitivity flow cell was used for detection. The column with pre-column
was Acquity UPLC HSS C18 SB 1.8 µm 2.1 × 100 mm (Waters Corp., USA). A gradient elution
was used to achieve chromatographic separation with a mobile phases A (water, containing 0.1%
formic acid and 10% acetonitrile) and B (acetonitrile, containing 2% water). The mobile phase in
the gradient elution progress was: 0–1 min 0% B, 1.5 min 1.7% B, 2.5 min 9.1% B, 7 min 17.6%
B, 8 min 28.3% B, 9–11 min 52.4% B, and 11.1–13 min 0% B. The flow rate was set at 0.4
mL/min and the column temperature was maintained at 40 °C. The auto-sampler temperature was
4 °C and the injection volume was 5 μL. The analytical run time for each sample was 13 min. UV
6

scan was obtained from 240 to 370 nm with resolution 0.6 nm and the trans-RSV concentration
was calculated as above..
2.4 Validation of analytical methods
UV/VIS spectroscopy, HPLC, and UPLC methods were validated in terms of specificity,
linearity, accuracy, precision, detection limit, and quantitation limit. The various validation
parameters and values for accepting the range of validation parameters were in accordance with
both ICH and FDA guidelines [26, 27].
Specificity was evaluated by measuring the blank solution, the trans-RSV standard (5 µg/mL), a
mixture of trans- and cis-RSV, and 85% or 7.5% of trans-RSV with degradation products in the
sample. The resulting mixtures were then analyzed for peak purity by scrutinizing UV/VIS
spectra from diode array detector. The mixture of trans- and cis-RSV was prepared from the 50%
methanol trans-RSV solution by exposure to UV light (366 nm) for 2 min at room temperature,
as was similarly described by Camont et al. [13]. The mixture of trans-RSV and its degradation
products were formed by trans-RSV degradation at alkaline pH. Resolution from the nearest
eluting peaks was determined according to ICH guidelines in the sample with a mixture of 85%
trans-RSV and degradation products [26].
Linearity was determined by calculating a regression line from the plot of the peak area versus
concentration for the seven standard solutions (0.5, 1, 2, 5, 7, 10, and 15 µg/mL) using the least
squares method.
Detection limit and quantitation limit were determined from their respective calibration curves
with equations described in ICH guidelines [26].
Accuracy was assessed using three concentration levels (1, 5, and 15 µg/mL) with three replicates
which were prepared by three different stock solutions and reported as average percent recovery
by the difference between the mean and the accepted true value.
Precision was assessed at two levels: repeatability and intermediate precision. Repeatability was
evaluated with concentrations 1, 5, and 15 µg/mL, which were injected three times into UPLC
system over short interval of time. Intermediate precision was evaluated by analyzing three
7

different standard samples (1, 5, and 15 µg/mL) on three different days. The results were reported
as the relative standard deviation (RSD).
2.5 Determination of trans-RSV chemical stability
Solutions with approximate 10 µg/mL of trans-RSV were prepared in buffers with pH 1.2, 6.8,
and 7.4 and stored at −22, 4, 25, and 37 °C for 90 days. At different time points the samples were
taken, filtered, and analyzed. Due to rapid degradation of trans-RSV at higher pH, stability
studies were performed a bit differently. Solutions with approximate 20 µg/mL of trans-RSV
were prepared in buffers with pH 8.0 (50 mM phosphate buffer), 9.0 (25 mM borate buffer), and
10.0 (50 mM carbonate buffer) stored at 37 °C. Samples (4 mL) were withdrawn and
immediately added to 4 mL of 50 mM HCl to stop the degradation process (final pH was below
4). The samples were analyzed by UV/VIS, HPLC, and UPLC method after filtration. The order
of the degradation reactions was determined with the graphic method [28]. Additionally, the
effect of temperature on the degradation rate of trans-RSV was characterized by the Arrhenius
equation:
ꢆ
ꢀ
ꢀꢁ ꢂ ꢃ ꢀꢁ ꢄ ꢅ
ꢇꢈ
where A is pre-exponential factor typical for trans-RSV degradation, Ea is activation energy
(J/mol), R is universal gas constant (8.314 J/Kmol), and T is the absolute temperature.
2.6 Determination of trans-RSV solubility at different pH
The solubility of trans-RSV was determined by adding an excess of trans-RSV into 100 mL
buffers with pH 1.2, 6.8, and 7.4. The suspensions were continuously stirred with a magnetic
stirrer at 200 rpm and 37 °C for 72 h. At three different time points (24, 48, and 72 h) samples
were withdrawn, immediately filtered, properly diluted and analyzed.
2.7 Preparation of nanofiber mats with trans-RSV and release study
Trans-RSV-loaded PCL nanofibers were prepared by electrospinning method. First, a 10% (w/w)
PCL solution was prepared by dissolving polymer and 0.03% (w/w) NaI in mixture of chloroform
8

and acetone in ratio 3:1 (w/w). Trans-RSV (5% w/w based on the mass of dry nanofibers) was
added to the PCL solution and mixed by Ultra Turrax T25 (Janke & Kunkel, IKA Labortechnik,
Germany) for 5 minutes at 15,000 rpm to obtain a homogenous solution. The electrospinning was
conducted at 15 kV, nozzle-to-planar collector distance 15 cm, and solution flow rate 1.3–2.5
mL/h resulted in nanofiber mats. The nanofiber morphology was examined using a 235 Supra
35VP-24-13 high-resolution scanning electron microscope (SEM) (Carl Zeiss,Germany) as
described by Rošic [29].
Trans-RSV release was performed on a fully calibrated dissolution apparatus using the paddle
method (USP Apparatus I, VanKel Dissolution Apparatus, model VK 7000, USA). 600 mL
buffers with pH 1.2, 6.8, and 7.4 were used as dissolution media. To prevent nanofiber floating,
102 2 mg mats were affixed to brackets that sank at the bottom of the vessel at the beginning of
the experiment. The temperature was maintained at 37 0.5 °C and vessel speed was kept at 50
rpm. At each sampling time point (0.25, 0.5, 1, 2, 3, 4, 8, and 12) 10 mL samples (not replaced)
of the dissolution medium were withdrawn, filtered, and immediately analyzed.
2.8 Statistical analysis
All experiments were performed at least in triplicate and the data are expressed as mean
standard deviation (S.D.). The results were statistically analyzed using repeated measured
analysis of variance (ANOVA) and one-way ANOVA, followed by Turkey’s post hoc test
(PRISM software package, Version 5, Graphpad Software Inc., San Diego, USA).
3 RESULTS
3.1 Validation of analytical methods
Validation of UV/VIS, HPLC, and UPLC methods was performed to ensure accurate, specific,
and reproducible results across the concentration range.
3.1.1 Specificity
The specificity of the selected analytical methods was evaluated on blank solution, standard
solution, the mixture of both isomers and trans-RSV with degradation products. Blank solution
9

did not show any peaks or interference on the spectrum or on the chromatograms. Trans-RSV
standard substance in solution eluted as a single peak at retention times 6.2 and 7.0 min for HPLC
and UPLC, respectively (Fig 1). The spectrum of trans-RSV standard was the same in all three
methods with the peak varying from 304 to 306 nm.
FIGURE 1 – 2 columns
The trans-RSV exposure to UV light resulted in isomerization. Its absorption maximum
decreased at 306 nm and a new peak increased at 287 nm, representing the maximum of cis-RSV
with time (Fig. 2A). The sample containing both isomers measured by all three methods is
presented in Fig. 1. Cis-RSV eluted later than trans-RSV, with retention times 8.8 min in HPLC
and 7.7 min in UPLC, due to its more lipophilic nature. The resolution between both peaks was
11.75 for HPLC and 7.5 for UPLC, which exceeded the required resolution (1.5). Extracted
chromatographic peaks clearly showed that cis-RSV also absorbs at 306 nm. Therefore, the
UV/VIS spectrum contained the sum of absorbances of both isomers, whereas the contribution
absorbance of each isomer to the spectrum cannot be determined precisely.
FIGURE 2 – 1 column
By contract, the increased media pH caused trans-RSV degradation, and the process with time is
presented in the UV/VIS spectra (Fig. 2B). When trans-RSV degraded, its maximum also
decreased and absorbance increased at 265 nm, showing degradation compounds. The formation
of degradation product was confirmed by HPLC and UPLC (Fig. 1). The two main degradation
products had lower retention times due to more hydrophilic nature and absorbed at 268 and 274
nm for HPLC, and at 267 and 279 nm for UPLC, which is in line with new appearing peak in
spectrum obtained by UV/VIS. However, as is seen in this spectrum, there was not possible to
determine the contribution of degradation products in mixture with trans-RSV in absorption
curve. Consequently, UV/VIS method was not enough specific, whereas both chromatographic
methods match the required specificity (HPLC resolution was 3.5 and UPLC 2.7, required 1.5).
10

Although, more and better separated peaks were observed on the chromatograms of UPLC
compared to HPLC. Thus, the UV/VIS method does not enable the separation of trans-RSV from
cis-RSV and degradation products and due to their contribution to the absorption at 306 nm the
HPLC or UPLC methods are advised to be used.
3.1.2 Linearity and sensitivity
Across the selected concentration range of trans-RSV in solution, a linear relationship between
the absorbance or the peak area and trans-RSV concentration was obtained by all three methods
with correlation coefficient (r) 1.0000 (Table 1), which is above the required value
(1.0000>0.999) [27]. The UV/VIS method was the most sensitive (Table 1).
TABLE 1
3.1.3 Accuracy and precision
Accuracy, presented as % of recovery, and precision, as repeatability and intermediate precision
in RSD, are shown in Table 2. Accuracy (98–102%), precision (< 1%), and intermediate
precision (< 2%) are within the acceptable limits for all analytical methods as required [27, 30].
The precision is better at higher concentration.
TABLE 2
3.2 Stability of trans-RSV at various pH levels and temperature
As evident from Fig. 3, trans-RSV degradation varies tremendously in aqueous solutions with
different pH levels. Trans-RSV was demonstrated to be relatively stable in acidic conditions,
whereas its degradation rate exponentially increases in alkaline ones. Results obtained by all
three analytical methods are similar only at pH 1.2, which means that most of trans-RSV is
relatively stable up to 90 days. At all higher pH levels it is clearly seen that HPLC and UPLC
show lower stability of trans-RSV than the UV/VIS, which reveals a smaller decrease of its
concentration. However, the results of both chromatographic methods are well-matched.
11

FIGURE 3 – 1 column
The calculated data for kinetics of trans-RSV degradation obtained at various pH levels,
correlation coefficients, and the half-lives are shown in Table 3. The trans-RSV degradation in
the pH range 1.2–8.0 followed first-order kinetics with a correlation coefficient higher than 0.99.
At pH 9.0 there was a combination of zero- and first-order kinetics, whereas at pH 10.0 trans-
RSV was stable for the first 2 min and then rapidly degraded with zero order. It is important to
point out that the half-life of trans-RSV is given in days for the pH range up to 7.4, in hours at pH
8.0, and in minutes at pH 9.0 (Table 3). For example, its half-life at pH 1.2 was 329 days, which
decreased to 3.3 minutes at pH 10. Trans-RSV in buffer with pH 9.0 was totally degraded in less
than 24 min and at pH 10.0 in less than 9 min (Fig. 3). The differences between the
chromatographic and UV/VIS methods increased with time due to the increased amount of
degradation products. For example, at after 8 days at pH 7.4 (Fig. 3) the concentrations obtained
by UV/VIS were highly inaccurate being approximately 700% higher as compared to HPLC or
UPLC. Consequently, the false UV/VIS data resulted in the lower calculated degradation
constants and the longer half-life of trans-RSV (Table 3).
TABLE 3
The stability of trans-RSV was high in the acidic conditions, whereas in the alkaline pH range of
6.8–9.0 the degradation constant increased exponentially. In the latter case it was found that the
pH dependence of the degradation rate constant k could be adequately described by an empirical
relation in the form of equation: ln(k) = 3.882pH − 32.685 (r = 0.9998) (Fig. 4).
FIGURE 4 – 1 column
The next study, the effect of temperature on trans-RSV stability in aqueous solutions with
various pH levels was monitored in the range of −22 to 37 °C for three months. At pH 7.4 trans-
RSV rapidly degraded at 25 and 37 °C, whereas the degradation was slowed at 4 °C and
prevented at −22 °C (Fig. 5). The kinetics of trans-RSV degradation at pH 7.4 and the
12

temperatures investigated followed first-order kinetics. The effect of temperature on the
degradation rate of trans-RSV was expressed by a linear plot of Arrhenius equation logkobs
=
14.063 − 4425(1/T) (r=0.97). The calculated activation energy of trans-RSV was 84.7 kJ/mol.
While not presented in detail, at different pH similar conclusions were derived. For instance,
trans-RSV degradation rate at 6.8 was lower at lower temperatures, e.g. a 1.2-fold decrease at 25
°C and a 22-fold decrease at 4 °C as compared to 37 °C. To conclude, in acidic media in which
trans-RSV was stable, temperature had the minimal impact on degradation. In contrast, at
alkaline pH higher temperatures further accelerated the trans-RSV degradation rate.
FIGURE 5 – 1 column
3.3 Trans-RSV solubility at different pH levels
Solubility of trans-RSV at pH 1.2 was 64 µg/mL, at pH 6.8 61 µg/mL, and at 7.4 50 µg/mL after
72 h, measured by UPLC (Table 4). The one-way ANOVA statistical test did not show any
significant difference between trans-RSV solubility at pH 1.2 and 6.8, but at pH 7.4 it was
significantly lower (p < 0.05) irrespective of the analytical method used. These results clearly
indicated that already weak alkaline pH affected trans-RSV solubility. The solubility data
obtained by different methods in the same time interval did not significantly differ at pH 1.2 and
6.8, but solubility at pH 7.4 was markedly different. UV/VIS data were significantly (p < 0.05)
higher for all three time points (by 10.3 %, 11.9 %, and 18.8 %, respectively) as compared to
UPLC results. These results were not only the consequence of solubility but also the
simultaneous degradation and lower specificity of the UV/VIS method.
TABLE 4
3.4 Release profiles of trans-RSV from nanofiber mats
Trans-RSV release profiles from nanofiber mats with 5% drug and an average nanofiber diameter
of 510 280 nm at various pH levels are presented in Fig. 6. Most of the trans-RSV was released
in the first 4 h and the remainder in the following 8 h in a sustained manner. The release profiles
13

at pH 1.2 and 6.8 were not dependent on pH or on the analysis method used. In the medium with
pH 7.4 different release profiles were obtained, because trans-RSV degradation took place
immediately after its release from the nanofibers. This was confirmed by appearance of new
peaks in the chromatograms. After 12 hours at pH 1.2 trans-RSV release reached 76.8%, at pH
6.8 80.1%, and at 7.4 pH it dropped to 48.6% (data obtained by UPLC). Comparison of release
profiles obtained by different analytical methods showed that the results obtained by UV/VIS
were found to be increasingly inaccurate the longer the experiment progressed.
FIGURE 6 – 2 columns
4 DISCUSSION
Previous studies have often reported the low stability and solubility of trans-RSV, but it was
rarely measured and discussed systematically. While previous study has reported that trans-RSV
is stable for months, except in high-pH buffers [12], our work shows that already at pH above 6.8
trans-RSV started to degrade significantly. Higher degradation rates were observed after 24 h in
buffers with pH 7.4 at 37 °C. This was also reported by other authors [31, 32]. Trans-RSV was
stable in acidic pH because its hydroxyl groups were protected from radical oxidation by
positively charged H3O+ [10]. On the contrary, trans-RSV was not stable in alkaline conditions.
Its degradation followed first order kinetics with reaction rates that increased exponentially in the
pH range 6.8–8.0 (Table 3). The degradation profiles above pH 8 were more complex and cannot
be described by simple first order kinetics. At pH 9 the degradation profile seemed to be
composed of two distinct regions with zero and first order kinetics and at pH 10 the degradation
profile seemed like an autocatalytic degradation process where some small amount of induction
time passes before the reaction started (Fig. 3 and Table 3). At pH 9.0 one hydroxyl group of
trans-RSV was predominantly deprotonated (depending on the study, pKa1 varies from 8.1 to 8.8.
[33]), whereas at pH 10.0 one hydroxyl group was fully deprotonated and another partially so
(pKa2 is 9.7–9.9 [33]). This suggests that the degradation mechanism depended on the degree of
dissociation of trans-RSV hydroxyl groups. The presence of phenate ions in the trans-RSV
structure made it particularly susceptible to oxidation due to electrophilic attack on this ion,
leading to the formation of the phenoxy radical [34]. Trans-RSV and its radical form led further
to the formation of several secondary degradation products excluding cis-RSV (Fig. 1).
14

Additionally, Callemien et al. reported that the presence of oxygen in the medium promoted
radical reactions leading to trans-RSV degradation [35]. The trans-RSV stability was also
dependent on the temperature. Degradation process exhibited intermediate sensitivity to
temperature, because its activation energy was within the range of 50–96 kJ/mol [36]. Therefore,
at 37 °C degradation was fast, whereas the freezing completely stopped the reaction by the
decreased molecular mobility. Consequently, its stability can be increased not only with a lower
pH but also with a lower temperature, as our findings confirmed (Fig. 5). To summarize, the
impact of individual factors appear in the following order (Table 5): UV light causes
isomerization of trans-RSV in minutes (Fig 2), pH effects differently on its degradation rate
(Table 3), and increased temperature enhance the degradation (Fig. 5). Thus the special attention
has to be dedicated to the experimental and storage conditions due to the low stability of trans-
RSV.
TABLE 5
Trans-RSV stability has been improved with special drug delivery systems or solid dosage forms.
For example, trans-RSV encapsulation in a nanoemulsion prevented oxidative degradation at 4
°C but was not effective at higher temperatures [9], and the stability of trans-RSV suspension
during 1 year of storage at pH 7.4 was improved by the addition of carboxymethylated (1,3/1,6)-
β‑ D‑ glucan [37]. When Jensen et al. evaluated crystalline trans-RSV according to ICH
guidelines for stability testing, they found that trans-RSV is stable for at least 3 months at
accelerated and ambient conditions even when exposed to UV and florescent light [38].
Therefore, crystalline trans-RSV or solid delivery systems with incorporated trans-RSV are the
most appropriate form from the stability point of view, as is also supported by our study using
nanofibers as an attractive novel drug delivery system [39].
The effect of pH on trans-RSV solubility was less pronounced as on stability (Table 4). The
solubility in buffers with pH 1.2 and 6.8 was approximately 60 µg/mL, whereas trans-RSV
solubility at pH 7.4 was 14% lower. This effect was due to lower trans-RSV stability at pH 7.4,
in which dissolution and degradation occurred at the same time, resulting in the increasing
concentration of degraded products which decreased trans-RSV solubility, probably due to
15

formation of insoluble complexes between them, as has already been shown for other drug
mixtures [40]. To the best of our knowledge, the reduction in trans-RSV solubility at pH 7.4 as
compared to 1.2 or 6.8 has not been published before. The trans-RSV solubility was determined
at 37 °C because this temperature is recommended for dissolution studies in sink conditions [25]
and for in vitro experiments on cell lines [41]. However, most studies reported trans-RSV
solubility at 25 °C to be 19.6–30 µg/mL [16, 42]. Solubility usually increases with temperature
[43], which could explain higher solubility determined in the study. However, other authors
reported trans-RSV solubility at 37 °C varying from 50 ng/mL to 20 µg/mL, which is not in line
with our results. Solubility data serve as a base for design of dosage forms and for decisions
regarding testing methodology. For example, the sink conditions for release studies cannot be
determined without knowing the solubility of the drug. To overcome low solubility in case of
some poorly soluble drug the addition of surfactants is sometimes necessary [25]. Das and Ng
claimed that trans-RSV solubility in simulated intestinal fluid was too low to observe its release
in standard dissolution apparatus even in the presence of a high amount of surfactant. Hence, they
devised an alternative method in which they estimated the amount of trans-RSV remaining in
their formulation after each time point instead of measuring the drug released in the medium [44].
On the contrary, our solubility data, release study, as well as results of several other researchers
demonstrates that the release studies of trans-RSV in different formulations can be normally
obtained in buffers without the addition of any surfactant [18, 20, 21, 45].
The optimization of electrospinning parameters enabled preparation of PCL nanofibers with
successfully incorporated trans-RSV without visible any crystals on the surface. The dominated
mechanism of trans-RSV release from PCL nanofibers was desorption from the polymer surface,
while the PCL nanofibers are not degrading at an appreciable rate, as was shown in other drug-
loaded PCL nanofibers [46]. The trans-RSV release was not affected by its degradation in acidic
medium. However, we determined significant difference at pH 7.4 and 37 °C after three hours
(Fig. 6), when the trans-RSV degradation started to become substantial (Fig. 3). To the best of
our knowledge, there are no published data reporting the problem of trans-RSV degradation
during release studies. For example, trans-RSV release profiles always increased with time with
no degradation effects exhibited in various published articles [18, 45, 47]. Only Sessa et al. 2014
reported degradation due to other reasons, including dissolved oxygen, temperature, and light
[21]. The possible explanation could be that the release rate dominated over the degradation rate,
16

as can be seen from our results. In the first 4 h, release was the dominant process, which can be
observed by the increased slope in the release profile. The subsequent sustained release was
slower compared to degradation kinetics resulting in the decreased trans-RSV concentration in
the medium (Fig. 6). Trans-RSV degradation can be an alternative interpretation to the reasons as
to why the release profiles of formulations did not reach 100% of released trans-RSV [45, 47].
Importantly, using the UV/VIS method for long releases at pH 7.4 and 37 °C results in falsely
higher concentrations and therefore in faster release profiles, which may mask trans-RSV
degradation. Consequently, published results obtained at these conditions may not present the
actual release profile [18].
Awareness of the high trans-RSV degradation rate in buffers with pH 7.4 is important also in
several other methods for in vitro evaluation of drug delivery systems. For example, cell cultures
are one of the most often used for studying the effect of trans-RSV on cells. Effect of trans-RSV
on cell proliferation assay can often be 48 hours long [48] or in some cases even 72 h [49].
During this time, more than half of trans-RSV at 37 °C and pH 7.4 would be degraded. There are
several other cell assays including a 24-hour or longer incubation of cells with free or trans-RSV-
loaded drug delivery systems for a purpose of evaluating its effect on the cell differentiation,
morphology, cytotoxicity, survival under stress conditions, antioxidant activity, and intracellular
reactive oxygen species levels [5, 41, 50]. Therefore, it is vital to realize that in the assays
conducted under these conditions the effect of trans-RSV would not be evaluated at its
predetermined concentration, but at its lower concentration and in the presence of degradation
products. This warning is also supported by findings from Long et al. and Yang et al. [31, 32],
where both confirmed the severe degradation of trans-RSV in a cell-buffered medium.
5 CONCLUSION
Validated UPLC method was developed and compared with HPLC and UV/VIS. Comparison of
the method specificity for trans-RSV and it’s degradation products followed in the order UPLC,
HPLC, and UV/VIS. Chromatographic methods are necessary for determining trans-RSV in
conditions under which it is not stable, because UV/VIS provides falsely higher concentrations
due low specificity. Our findings reveal that trans-RSV in a water solution was stable at room or
body temperature only under acidic conditions, whereas it degraded exponentially if the pH
became alkaline. Thus the stability of trans-RSV in liquid dosage form can be improved by
17

reducing the pH and temperature, and limiting exposure to oxygen and light. It is important to
keep in mind that trans-RSV dissolution and release studies done at pH 7.4 and 37 °C (as well as
other experiments done under similar conditions) could be accompanied by the presence of
significant trans-RSV degradation. This effect is more pronounced the longer the experiment. In
our case it was evident already after three hours. In this regard, it is worthwhile to note that
special attention has to be paid to trans-RSV dissolution testing or biological assays on cell lines
in order to get the relevant data. This study is of interest because it draws attention to the
importance of experimental conditions and their influence on the chemical stability of trans-RSV
and the implications this has for formulation development, storage, and analysis.
Acknowledgements
This research was financially supported by the Slovenian Research Agency (Program P1-0189
and Research Projects J1-6749).
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22

CAPTIONS
Fig. 1: UV/VIS, HPLC, and UPLC spectra and chromatograms of trans-RSV standard and
trans-RSV exposed to light or pH 9 resulting in isomerization and degradation, respectively. The
chromatograms of HPLC and UPLC were obtained at 306 nm. The spectra of two main
degradation products (red and green), trans-RSV (violet), and cis-RSV (orange) were extracted
from their retention times. (AU – absorbance units).
Fig. 2: The UV/VIS absorption spectra of (A) isomerization of trans-RSV to cis-RSV with UV
light at 366 nm after 0, 1, 2, 3, 4, 5, 7.5, and 15 min at room temperature and (B) degradation of
trans-RSV in buffer with pH 7.4 after 0, 1.5, 2.4, 8, 11, 21.5, 30.8, and 46.2 h at 37 °C.
Fig. 3: Kinetics of trans-RSV degradation dependent on pH levels. Concentrations of trans-RSV
at different time points are presented by symbols whereas curves present fitted degradation order
according to Table 3. Each point represents the mean of at least three measurements S.D.,
which is small; in many cases within the size of the symbols.
Fig. 4: Relationship between degradation kinetics log(k) of trans-RSV and pH aqueous solution
at 37 °C.
Fig. 5: Trans-RSV stability in an aqueous solution with pH 7.4 stored at different temperatures in
darkness for 3 months measured by UV/VIS, HPLC, and UPLC. Each point represents the mean
of at least three measurements S.D., which is small, in many cases within the size of the
symbols.
Fig. 6: SEM image of 5% trans-RSV-loaded nanofibers and its release profiles in buffer with pH
1.2, 6.8, and 7.4 at 37 °C using UV/VIS, HPLC, and UPLC methods.
23

TABLES
Table 1: Detection limit (DL), quantitation limit (QL), and linearity parameters for UV/VIS,
HPLC, and UPLC methods.
Linear
response
range
Linearity
Slope
DL
QL
Method
(µg/mL) (µg/mL)
r
(µg/mL)
UV/VIS
HPLC
UPLC
0.09
0.25
0.14
0.26
0.77
0.43
0.5–15
0.5–15
0.5–15
0.1268 1.00000
105.0 0.99998
216119 0.99996
24

Table 2: Accuracy and precision for trans-RSV determined by UV/VIS, HPLC, and UPLC.
Precision
Repeatability Intermediate
Nominal
conc.
(µg/mL)
Accuracy
(%)
Method
(%)
0.85
0.47
0.26
precision (%)
1
5
15
99.3
99.5
99.8
1.36
1.11
1.03
UV/VIS
1
5
15
100.9
99.1
98.7
0.75
0.40
0.33
2.00
1.31
0.53
HPLC
UPLC
1
5
15
101.2
101.0
100.8
0.58
0.17
0.05
1.27
0.08
0.48
25

Table 3: The kinetics of trans-RSV degradation obtained at various pH levels with UV/VIS,
HPLC, and UPLC (k - degradation constant, R - Pearson's correlation coefficient, t1/2 - half-life).
k (µM/h (zero order), /h (first order))
R
t1/2
HPLC
303 days
pH Reaction order
UV/VIS
-0.000532
-0.00125
-0.0099
-0.103
-246
HPLC
-0.000863
-0.00192
-0.0178
-0.176
-315
UPLC
-0.000977
-0.00199
-0.0179
-0.186
-328
UV/VIS HPLC UPLC
UV/VIS
UPLC
1.2
6.8
7.4
8.0
9.0
0.8093
0.9987
0.9822
0.9955
0.9983
0.9926
0.9955
0.9876 0.9227
0.9994 0.9992
0.9969 0.9961
0.9993 0.9991
0.9998 0.9997
0.9952 0.9966
0.9998 1.0000
first
first
first
first
zero
441 days
329 days
23.2 days 15.1 days 14.5 days
2.9 days
6.7 h
10.1 min
10.9 min
4.3 min
1.6 day
3.9 h
7.7 min
4.1 min
3.3 min
1.6 day
3.7 h
7.7 min
4.2 min
3.3 min
first
-3.82
-537
-10.12
-675
-9.95
-716
10.0
zero after 2 min
26

Table 4: Trans-RSV solubility (µg/mL) in buffers with pH 1.2, 6.8, and 7.4 at 37° C measured
by UV/VIS, HPLC and UPLC.
Solubility (µg/mL)
Time
(h)
pH
UV/VIS
HPLC
UPLC
24
48
72
60.8 1.0 60.2 1.2 61.2 1.8
61.9 2.2 62.6 2.2 62.7 1.2
64.2 1.2 63.7 1.1 64.3 1.4
1.2
24
48
72
61.9 0.4 61.4 0.3 61.0 1.3
62.2 0.2 61.9 0.3 62.3 1.1
62.4 1.1 60.8 1.4 61.2 2.1
6.8
7.4
24
48
72
58.9 0.9 54.1 0.7 53.4 1.5
58.8 0.9 52.8 1.5 52.5 1.1
59.5 1.4 51.0 1.1 50.1 0.7
27

Table 5: The impact of the individual factors to trans-RSV isomerization or degradation to its
half-life.
Factor
Process
Degradation time
Reference
Fig. 2
UV light (366 nm) isomerization a few minutes
pH 9 or 10
pH 8
degradation
degradation
a few minutes
hours
Fig. 3, Table 3
Fig. 3, Table 3
[13]
sunlight
pH 7.4
isomerization hours
degradation
degradation
degradation
degradation
a few days
more days
Fig. 3, Table 3
Fig. 3, Table 3
pH 6.8
↓temperature
pH 1.2
decrease degradation rate Fig. 5
almost a year Fig. 3, Table 3
28