The effects of Tween-80 on the integrity of solutions of capsaicin: useful information for performing tussigenic challenges.

Abstract

:
Because variable results of capsaicin challenges may be due to the incomplete solubility of capsaicin, we sought to determine if the use of Tween-80 in solutions of capsaicin improves actual concentrations of freshly prepared and stored solutions.
Capsaicin solutions ranging from 0.5-128 muM were mixed with and without Tween-80. Samples of various concentrations were then stored under 4 environmental conditions: 4 degrees C, protected from light; room temperature, protected from light; room temperature, exposed to light; -20 degrees C. All samples were analyzed initially, and at 2 and 4 months.
While freshly prepared solutions with Tween-80 had consistently higher concentrations than those prepared without Tween-80 (83% vs. 69%), Tween-80 does not facilitate complete solubility. For solutions stored at 4 degrees C and protected from light, there was a significant decrease after 2 months in low concentration solutions of both the Tween-80 and non-Tween-80 solutions. Both Tween-80 and non-Tween-80 containing solutions significantly decreased in concentration after 2 months when stored at room temperature and protected from light, room temperature and exposed to light, and -20 degrees C. Concentrations of solutions made of 4 muM or higher are stable when stored at 4 degrees C and protected from light for 4 months.
While the inherent difficulty of forcing capsaicin into solution cannot be eliminated, it can be improved with Tween-80. However, the addition of Tween-80 does not prevent the breakdown of stored capsaicin solutions. We recommend preparing and storing capsaicin solutions according to the methods and results of this study.

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BioMed Central
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Cough
Open Access
Research
The effects of Tween-80 on the integrity of solutions of capsaicin:
useful information for performing tussigenic challenges
Scott E Kopec*†1, Richard S Irwin†1, Ronald J DeBellis†2, Mark B Bohlke†2
and Timothy J Maher†2
Address: 1The Division of Pulmonary, Allergy, and Critical Care Medicine, UMASS Memorial Medical Center, Worcester, MA, USA and
2Massachusetts College of Pharmacy and Health Sciences, Boston, MA, USA
Email: Scott E Kopec* - kopecs@ummhc.org; Richard S Irwin - irwinr@ummhc.org; Ronald J DeBellis - rjdebellis@wor.mcphs.edu;
Mark B Bohlke - mark.bohlke@bos.mcphs.edu; Timothy J Maher - timothy.maher@bos.mcphs.edu
* Corresponding author †Equal contributors
Abstract
Background: Because variable results of capsaicin challenges may be due to the incomplete
solubility of capsaicin, we sought to determine if the use of Tween-80 in solutions of capsaicin
improves actual concentrations of freshly prepared and stored solutions.
Methods: Capsaicin solutions ranging from 0.5–128 μM were mixed with and without Tween-80.
Samples of various concentrations were then stored under 4 environmental conditions: 4°C,
protected from light; room temperature, protected from light; room temperature, exposed to
light; -20°C. All samples were analyzed initially, and at 2 and 4 months.
Results: While freshly prepared solutions with Tween-80 had consistently higher concentrations
than those prepared without Tween-80 (83% vs. 69%), Tween-80 does not facilitate complete
solubility. For solutions stored at 4°C and protected from light, there was a significant decrease
after 2 months in low concentration solutions of both the Tween-80 and non-Tween-80 solutions.
Both Tween-80 and non-Tween-80 containing solutions significantly decreased in concentration
after 2 months when stored at room temperature and protected from light, room temperature and
exposed to light, and -20°C. Concentrations of solutions made of 4 μM or higher are stable when
stored at 4°C and protected from light for 4 months.
Conclusion: While the inherent difficulty of forcing capsaicin into solution cannot be eliminated,
it can be improved with Tween-80. However, the addition of Tween-80 does not prevent the
breakdown of stored capsaicin solutions. We recommend preparing and storing capsaicin solutions
according to the methods and results of this study.
Backround
Inhalational challenges using solutions of capsaicin have
been used to determine cough thresholds in subjects [1],
and to test the effectiveness of antitussive agents [2]. How-
ever, several studies have demonstrated varying results of
capsaicin tussigenic challenges [3,4]. We demonstrated in
a prior study [5] the incomplete solubility of capsaicin in
ethanol/saline solution, and the instability of stored cap-
saicin solutions. It is possible that these findings are
potential sources of the variable results. We also showed
Published: 27 May 2008
Cough 2008, 4:3doi:10.1186/1745-9974-4-3
Received: 9 January 2008
Accepted: 27 May 2008
This article is available from: http://www.coughjournal.com/content/4/1/3
© 2008 Kopec et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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that solutions less than 4 μM decreased in concentration
after 2 months when stored in the dark at 4°C, while all
concentrations stored at room temperature, whether pro-
tected from light or not, decreased in concentration after
4 months of storage.
While Tween-80 has been utilized by others [6-9] when
making solutions of capsaicin in an empiric attempt to
increase the solubility of capsaicin, these authors did not
measure the actual concentrations of the solutions.
Tween-80 (Sorbitan mono-9-octadecenoate poly(oxy-1.2-
ethanediyl), also known as polysorbate 80, is an agent
that is used as an emulsifier and dispersing agent for med-
ications designed for internal use. It is very soluble in
water, alcohol, and organic solvents such as toluene. Con-
sequently, we sought to prospectively and objectively
determine, for the first time, if the use of Tween-80 in
solutions of capsaicin improves the actual concentration
of freshly prepared solutions of capsaicin, and to deter-
mine if Tween-80 could result in maintaining the concen-
trations of stored solutions kept under various
environmental conditions. Because capsaicin tussigenic
challenges are being used more frequently to assess the
sensitivity of the cough reflex and the efficacy of antitus-
sive agents [10], it is clinically important to standardize all
aspects of the capsaicin tussigenic challenge methodol-
ogy.
Methods
Two stock solutions of 128 μM concentrations of capsai-
cin were made. Solution 1 (without Tween-80) was made
by dissolving 39 mg of capsaicin powder (Sigma Chemi-
cal Co., St Louis, MO) in 12.5 mL of 100% ethanol; this
was then diluted with 0.9% sodium chloride solution to a
total volume of 1 liter. Solution 2 (with Tween-80) was
made by dissolving 39 mg of capsaicin powder in 5 mL of
100% ethanol. To this, 5 mL of Tween-80 (Fisher Scien-
tific, Leicestershire, UK) was then added, and the solution
was diluted with 0.9% sodium chloride solution to a total
volume of 1 liter.
The two stock solutions of 128 μM concentration of cap-
saicin were repeatedly diluted 1:1 with 0.9% sodium chlo-
ride solution to make 2 sets of solutions of 64, 32, 16, 8,
4, 2, 1, and 0.5 μM. One set contained Tween-80 (from
stock Solution 2) and the other set did not contain Tween-
80 (from stock Solution 1). Aliquots of these freshly made
solutions were then analyzed for actual concentration.
Additional aliquots of each of the freshly prepared solu-
tions, both with Tween-80 and without Tween-80, were
separated for storage under several environmental condi-
tions. Samples of all nine of the concentrations of both
the Tween-80 and non-Tween-80 solutions were stored at
4°C and protected from light. Samples of solutions of 4
μM, 8 μM, and 16 μM capsaicin concentrations were
stored at room temperature and protected from light, and
room temperature exposed to light. Finally, samples of
solutions of 32 μM, 64 μM, and 128 μM were stored in a
freezer at -20°C. Samples to be kept in the dark (at room
temperature, refrigerated at 4°C, and frozen at -20°C)
were placed in amber vials, and the vials were then stored
in light protecting boxes. Samples to be stored exposed to
light were placed in clear vials and stored on a laboratory
shelf. Samples were tested at baseline and then every 2
months for a total of 4 months. During testing, each sam-
ple was tested twice. The concentration of each individual
sample was determined by calculating the average of the 2
tests. However, if the results of the two tests on any one
solution differed by more than 3%, they were discarded
and two additional tests were performed to obtain data
within the acceptable variations.
The concentrations of the solutions of capsaicin were
measured by high performance liquid chromatography
(HPLC). Measurements were made both via electrochem-
ical detection and ultraviolet detection. Initial calibration
tests demonstrated that electrochemical detection pro-
duced non-linear results for concentrations above 16 μM.
Therefore, electrochemical detection was used only on the
solutions of 16 μM or less, and ultraviolet detection was
used for the 32 μM, 64 μM, and 128 μM concentrations.
The HPLC system consisted of an ESA 540 autosampler
(ESA, Inc., Chelmsford, MA), an ESA 580 pump, and a
Spectra-Physics SP8490 UV detector (ThermoFinnigan,
Waltham, MA) set at 280 nm. Data were acquired and
analyzed by PC/Chrom+ software (H&A Scientific, Green-
ville, NC). The column was a Phenomenex Luna (C18, 150
× 4.6 mm, 5 μm particles, 100 Å pores) (Torrance, CA).
The method was isocratic, with a flow rate of 1.5 mL/min
and a run time of 12 minutes. Injection volume was 20
μL. Capsaicin eluted at 5 minutes.
The HPLC mobile phase consisted of 3 g of NaH2PO4 dis-
solved in 480 mL of purified H2O, to which 520 mL ace-
tonitrile was added. This solution was adjusted to pH 4
with H3PO4 and filtered through a 0.2 μm nylon mem-
brane prior to use. The mobile phase was not re-circulated
because it was found that the introduction of Tween-80
significantly increased the retention time of capsaicin after
repeated injections.
For statistical analysis, solutions stored at 4°C and pro-
tected from light were divided into "low concentration
solutions" which included the 0.5, 1.0, and 2.0 μM con-
centrations, and "high concentration solutions" which
included the 4.0, 8.0, 16.0, 32.0, 64.0, and 128.0 μM con-
centrations. Statistical analysis of the data was performed
using paired t-tests and one-way

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ANOVA. Calculations were performed using SSPS-12 soft-
ware (SSPS Inc., Chicago, IL). The 0.05 level of signifi-
cance was used throughout.
Results
Appearance of freshly prepared solutions
The initial stock solution containing Tween-80 was clear
in color, while the non-Tween containing stock solution
had a whitish, cloudy appearance. The subsequent con-
centrations of the non-Tween containing solutions
became less cloudy with further dilution with saline as
described in the Methods Section. The Tween-containing
solutions remained clear even after further dilution.
Results of baseline freshly prepared solutions
Predicted and actual initial concentrations of Tween-80
and non-Tween-80 containing solutions are summarized
in Table 1. For each of the paired concentrations, those
containing Tween-80 were consistently higher. The 128
μM stock solution of capsaicin prepared with Tween-80
had an actual baseline concentration of 120.5 μM, while
the 128 μM stock solution prepared without Tween-80
had an actual baseline concentration of 92.7 μM. The
average baseline concentration of all the freshly prepared
solutions of capsaicin prepared with Tween-80 was 83.9 ±
6.8% of predicted, with a range of 74.4–94.1%. The aver-
age baseline concentration of the freshly prepared solu-
tions without Tween-80 was 63.4 ± 3.4% with a range of
61.5–72.4%. The actual concentrations of all the Tween-
80 containing solutions were significantly greater than the
non-Tween-80 solutions (p < .001).
Results of stored solutions containing Tween-80
Solutions stored at 4°C and protected from light
These data are summarized in Table 2. For individual con-
centrations of Tween-80 containing solutions stored at
4°C and protected from light, there was a significant
decrease in the lower concentrations (0.5, 1, and 2 μM)
compared to the higher concentrations (4, 8, 16, 32, 64,
and 128 μM) after 2 months (p = .041). The low concen-
tration solutions averaged a 20.1% decrease in concentra-
tion after 4 months while the high concentration
solutions had a slight increase in average concentration of
1.2% after 4 months. The largest decrease in concentra-
tion was seen in the 0.5 μM solutions.
Solutions stored at room temperature and protected from light
These data are summarized in Table 3. All the Tween-80
solutions stored at room temperature and protected from
light had a significant drop in concentration after 2
months (p < .001) and 4 months (p < .001) compared to
baseline concentrations.
Solutions stored at room temperature and exposed to light
These data are summarized in Table 3. Similarly, all
Tween-80 containing solutions stored at room tempera-
ture and exposed to light had a significant drop in concen-
tration at 2 months (p = .032) and 4 months (p = .042).
Solutions stored at -20°C
These data are summarized in Table 4. All the Tween-80
containing solutions had a significant decrease in concen-
tration after 4 months when stored at -20°C and protected
from light (p < .001) when compared to baseline concen-
trations.
Results of stored solutions without Tween-80
Solutions stored at 4°C and protected from light
These data are summarized in Table 2. For solutions
stored at 4°C and protected from light, there were signifi-
cant decreases in the low concentration solutions com-
pared to the high concentration solutions after 2 months
(p = .012). After 4 months, the low concentration solu-
tions averaged a 7.4% decrease in concentration, while
the high concentration solutions decreased an average of
7.6%. The largest decreases were seen in the 0.5 μM
(13.9% decrease) and 128 μM (31.3% decrease) solu-
tions.
Solutions stored at room temperature and protected from light
These data are summarized in Table 3. All the non-Tween-
80 solutions stored at room temperature and protected
from light had a significant drop in concentration after 2
months (p < .001) and 4 months (p < .001) compared to
baseline concentrations.
Solutions stored at room temperature and exposed to light
These data are summarized in Table 3. Significant
decreases in concentration were also seen in the solutions
without Tween-80 that were stored at room temperature
and exposed to light at 2 months (p = .002) and 4 months
(p = .013) compared to baseline.
Table 1: Predicted and Actual Initial Concentrations of Tween-
80 vs. non-Tween-80 Containing Solutions.
Actual Concentrations (% predicted)
Predicted Concentration (in μM)Tween-80non-Tween-80
0.5
1.0
2.0
4.0
8.0
16.0
32.0
64.0
128.0
0.44 (88.4)
0.78 (78.1)
1.49 (74.4)
3.12 (78.0)
6.27 (78.4)
13.74 (85.9)
28.54 (89.2)
56.90 (88.9)
120.45 (94.1)
0.33 (67.4)
0.64 (63.5)
1.24 (62.2)
2.48 (62.0)
4.92 (61.5)
10.37 (63.1)
21.82 (68.2)
42.56 (66.5)
92.67 (72.4)

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Solutions stored -20°C
These data are summarized in Table 4. All the solutions
without Tween-80 stored at -20°C and protected from
light had significant decreases in concentration at 4
months compared to baseline (p < .001).
Comparative results of Tween-80 vs. non-Tween-80 stored
solutions
Solutions stored at 4°C and protected from light
For the solutions stored at 4°C and protected from light,
there was a significant drop in the concentration of the
non-Tween-80 containing solutions when compared to
the Tween-80 solutions at 2 and 4 months (p = .003)
(Table 2).
When comparing the lower concentrations (0.5 μM and 1
μM) of solutions with and without Tween-80 in Table 2,
the trend of the data suggest that the non-Tween-80 con-
taining solutions of these 2 concentrations might be more
stable after 4 months of storage than the Tween-80 con-
taining solutions. However, this trend was not found to be
significant (p = .189).
Solutions stored at room temperature and protected from light
While there were significant decreases in all solutions
stored at room temperature and protected from light, the
decrease in the non-Tween-80 containing solutions was
significantly greater than the Tween-80 containing solu-
tions at 2 and 4 months (p < .001) (Table 3).
Solutions stored at room temperature and exposed to light
Identical results were seen with all solutions stored at
room temperature and exposed to light. Whether contain-
ing Tween-80 or not, there were significant decreases in all
concentrations at 2 months (p = .001) and 4 months (p <
.001) when compared to the baseline concentration
(Table 3). The decreases in concentrations of the non-
Tween-80 containing solutions were greater at 2 months
(p < .001) and 4 months (p < .001) than the decreases in
the Tween-80 containing solutions.
While all concentrations of solutions stored at room tem-
perature significantly decreased at 2 and 4 months com-
pared to baseline, the decreases were greater in the
solutions exposed to light vs those protected from light (p
< .001 at 2 and 4 months).
Table 2: Actual Concentrations of Tween-80 and non-Tween-80 containing solutions at 0, 2, and 4 months stored at 4°C and protected
from light.
Actual Concentration (% predicted)
Tween-80 Non-Tween-80
Predicted Concentration (in μM)0 month 2 months 4 months0 month2 months 4 months
0.5
1.0
2.0
4.0
8.0
16.0
32.0
64.0
128.0
0.44 (88.4)
0.78 (78.1)
1.49 (74.4)
3.12 (78.0)
6.27 (78.4)
13.74 (85.9)
28.54 (89.2)
56.90 (88.9)
120.45 (94.1)
0.23 (45.4)
0.59 (58.9)
1.30 (64.9)
2.97 (74.3)
5.94 (74.3)
11.76 (73.5)
26.50 (82.8)
56.19 (87.8)
115.07 (89.9)
0.24 (47.8)
0.59 (58.6)
1.47 (73.4)
3.32 (83.0)
7.10 (88.8)
12.62 (78.9)
27.30 (85.3)
55.87 (87.3)
119.68 (93.5)
0.33 (67.4)
0.64 (63.5)
1.24 (62.2)
2.48 (62.0)
4.92 (61.5)
10.37 (63.1)
21.82 (68.2)
42.56 (66.5)
92.67 (72.4)
0.24 (47.8)
0.55 (54.5)
1.06 (53.0)
2.36 (59.1)
4.37 (54.6)
8.32 (52.0)
21.42 (66.9)
40.32 (63.1)
43.26 (33.8)
0.27 (53.5)
0.58 (58.2)
1.18 (59.2)
2.49 (62.2)
4.74 (59.3)
8.99 (56.2)
21.37 (66.8)
42.37 (66.2)
52.61 (41.1)
Table 3: Actual Concentrations of Tween-80 and non-Tween-80 containing solutions at 0, 2, and 4 months stored at room
temperature and protected from light, and room temperature exposed to light.
Actual Concentration (% predicted)
Tween-80 Non-Tween-80
Predicted Concentrations (in μM) Environment0 month2 months 4 months0 month2 months4 months
4.0
8.0
16.0
4.0
8.0
16.0
RT/dark
RT/dark
RT/dark
RT/light
RT/light
RT/light
3.12 (78.0)
6.27 (78.4)
13.74 (85.9)
3.12 (78.0)
6.27 (78.4)
13.74 (82.7)
1.24 (31.1)
3.93 (49.1)
8.93 (55.8)
0.13 (3.2)
0.74 (9.2)
3.44 (21.5)
0.79 (19.8)
3.47 (43.4)
8.46 (52.9)
0.03 (0.7)
0.70 (8.7)
1.73 (10.8)
2.48 (62.0)
4.92 (61.5)
10.37 (63.1)
2.48 (62.0)
4.92 (61.5)
10.37 (63.1)
0.45 (11.2)
1.06 (13.3)
3.62 (22.6)
0.08 (2.0)
0.30 (3.7)
2.70 (16.9)
0.04 (1.1)
0.61 (7.6)
1.47 (9.2)
0.00 (0.0)
0.00 (0.0)
1.12 (7.0)
RT/dark = room temperature and protected from light, RT/light = room temperature and exposed to light

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Solutions stored at -20°C
When compared to the baseline concentration, both the
Tween-80 containing solutions and those without Tween-
80 had a significant decrease in concentration after 4
months when stored at -20°C and protected from light (p
< .001). There was no significant difference between the
decrease in concentration of the Tween-80 compared to
the decrease in the non-Tween-80 containing solutions
after 4 months (p = .136).
Discussion
From our study, two notable findings emerged that have
not been previously reported regarding the use of Tween-
80 in preparing capsaicin solutions. First, our results dem-
onstrate that the use of Tween-80, an emulsifier and dis-
persing agent, significantly improved the actual
concentration of freshly prepared solutions of capsaicin
compared to solutions not containing Tween-80. Second,
the use of Tween-80 did not prevent solutions from
decreasing in concentration when stored at room temper-
ature and protected from light, room temperature and
exposed to light, or frozen at -20°C after 4 months. This
trend is consistent with the results of our first study in
which we analyzed only solutions made without Tween-
80 [5].
With respect to the use of Tween-80 to prevent the break-
down of capsaicin solutions over time, when stored at var-
ious environmental conditions, we found that solutions
of 2 μM and less of both the Tween-80 containing and
non-Tween-80 containing solutions did significantly
decrease in concentration after 2 months compared to the
higher concentration. However, it did appear that the
higher concentration of Tween-80 containing solutions
were more stable after 4 months when stored at 4°C and
protected from light compared to the non-Tween-80 solu-
tions. The non-Tween-80 data in this study are similar to
the results in our initial study of non-Tween-80 contain-
ing solutions [5]. Although the actual, initial concentra-
tions of the freshly prepared non-Tween-80 solutions
were lower in this study compared to our first [5], we
attribute the difference to our using a newer assay in this
study. After testing both assays in the laboratory, we
found that the assay used in this current study provided
more consistent results. Despite the differences in the val-
ues between our two studies, the overall results with
regard to incomplete solubility and the breakdown of the
stored solutions were similar. Because the serial dilutions
of the freshly prepared solutions of capsaicin consistently
resulted in concentrations nearly half of the previous solu-
tion's concentrations (Table 1) and this finding appeared
to be independent of whether or not Tween-80 was used
in the initial stock solution, it appears that the major ben-
efit of using Tween-80 when preparing the solutions
appears to be the increase in concentration of the initial
stock solution.
Tween-80 is commonly used in pharmaceutical products
to improve solubility of water insoluble medications.
While Tween-80 is felt to be safe for intravenous and
inhaled use, there have been a few reported cases of toxic-
ity and hypersensitivity that have been associated with
Tween-80 found in some medication preparations. Liver
and kidney damage in infants who received intravenous
vitamin E has been attributed to Tween-80 [11], as has
severe acute hepatitis seen in patients receiving intrave-
nous amiodarone [12]. While we are not aware of any
reported toxicities associated with the inhaled use of
Tween-80, there is a case report of contact dermatitis that
was felt to have developed as a result of repeated exposure
to Tween-80 found in budesonide solution [13]. Never-
theless, a retrospective analysis of 122 studies containing
a total of 4833 subjects provides some evidence that cap-
saicin cough challenges in humans are safe [14], but it is
unclear how many of these studies used Tween-80 in the
preparation of the capsaicin solutions and how good data
capture was.
Methodological considerations
While there have been several reported methods for mak-
ing capsaicin solutions without Tween-80 [1,2,5], we are
aware of only one other published reported method using
Tween-80 [6-9]. In all 4 of these studies by Fujimura et al,
the identical method was used to prepare the capsaicin
solutions with Tween-80, but capsaicin solubility was not
objectively assessed. Our method for making solutions of
capsaicin with Tween-80 differed slightly from the
method used by Fujimura et al. Specifically, Fujimura et al
Table 4: Actual concentrations of Tween-80 and non-Tween-80 containing solutions at 0 and 4 months stored at -20°.
Actual Concentration (% predicted)
Tween-80 Non-Tween-80
Predicted Concentration (in μM)0 Month4 Months0 Month4 Months
32.0
64.0
128.0
28.54 (89.2)
56.90 (88.9)
120.45 (94.1)
22.62 (70.7)
47.81 (74.7)
104.32 (81.5)
21.82 (68.2)
42.56 (66.5)
92.67 (72.4)
15.87 (49.6)
30.53 (47.7)
69.63 (54.4)