Pharmacokinetic Profile of Astaxanthin Nanoemulsion Using HPLC
(High-Performance Liquid Chromatography) With Oral Routes
Lusi Nurdianti*1, Fajar Setiawan1, Taofik Rusdiana2, Iyan Sofyan2, Elis
Lisnawati1, Ardianes Firmansya1
1Faculty of Pharmacy, Bakti Tunas Husada University, Tasikmalaya 46115, Indonesia.
2Faculty of Pharmacy, Padjadjaran University, Sumedang 45363, Indonesia.
Submitted: 17/01/2024, Revised: 23/05/2024, Accepted: 20/12/2024, Published: 24/01/2025
ABSTRACT
Astaxanthin is a very strong antioxidant of the xanthophyll carotenoid group with
very lipophilic properties, so in oral administration, its bioavailability is very low.
This study aimed to examine the differences in the pharmacokinetic profile of
astaxanthin and astaxanthin nanoemulsion via the oral route using the HPLC
method. Research methods include validation methods using HPLC and
continued determining pharmacokinetic profiles to determine whether
astaxanthin in plasma is validated. The plasma was deproteinized with ethanol:
tetrahydrofuran (1: 9), mobile phase Methanol: water milliQ: ethyl acetate
(82:8:10 (v/v)), the flow rate is 1.2 mL/min, wavelength 474 nm and the sample
measured isocratic. Calibration curves 0.25-10 mg/L,% Recovery 102.92, 103.11
and 102.25,% RSD 1.33, 1.65 and 2. The HPLC method is fast, simple, and can
be used for routine analysis of astaxanthin. The results showed that in the
astaxanthin nanoemulsion, there was an increasing in Cmax and AUC0-∞ which
affected increasing the bioavailability value. Therefore, it was concluded that the
study showed the presence of astaxanthin in nanoemulsions has greater
absorption power than pure astaxanthin.
Keywords: Pharmacokinetic profile, Astaxanthin, Validation of the HPLC
method, Oral route
Vol. 5, Issue 2, 2023 (441-448)
http://journal.unpad.ac.id/IdJP
*Corresponding author,
e-mail: lusinurdianti83@gmail.com L. Nurdianti )
https://doi.org/10.24198/idjp.v5i2.52645
© 2023 L. Nurdianti et al
442
1. Introduction
The objectives and hypothesis of
the study should be clearly stated.
Astaxanthin is a carotenoid xanthophyll
with powerful antioxidants (Fassett &
Coombes, 2011). Astaxanthin has higher
antioxidant activity when compared to
various carotenoids such as lutein,
lycopene, and carotene (Naguib, 2000).
The limited dissolution of astaxanthin in
gastrointestinal fluids is one of the causes
of its low bioavailability. Another
contributing factor is the saturation of
carotenoids to enter the micelles formed
by bile salts in the gastrointestinal tract at
high doses (Domínguez-Hernández et al.,
2016; Fassett, R. G., & Coombes, J. S.,
2011).
Nanoemulsions can increase
solubility in water, improve permeation
due to their adhesive properties, prevent
enzymatic degradation in the intestinal
wall and liver, and protect against
gastrointestinal degradation to increase
stability and bioavailability (Ragelle et
al., 2012; Reyes-Munguía, Azúara-Nieto,
Beristain, Cruz-Sosa, & Vernon-Carter,
2009; Shrewsbury, Bosco, & Uster, 2009;
Sozer & Kokini, 2009). The oral route is
one of the most widely used because of
its easy, practical, and convenient
application. (Khan, Boateng, Mitchell, &
Trivedi, 2015). The development of
astaxanthin in nanoemulsions is expected
to increase the solubility, dissolution,
absorption, and bioavailability of
lipophilic drugs through pharmacokinetic
testing and parameter determination.
In humans, the bioavailability of
carotenoids is low and varies from 10 to
50% of the administered dose due to their
low solubility in digestive tract fluids,
leading to poor absorption (Nagao, 2011).
Astaxanthin is a highly lipophilic
compound with low oral bioavailability
due to first-pass metabolism. Low
bioavailability means low plasma drug
levels (Choi, Kang, Yang, Lee, & Shin,
2011). However, the bioavailability of
astaxanthin can be increased by
introducing the drug into an emulsion
form (Odeberg, Lignell, Pettersson, &
Höglund, 2003).
This study was conducted to test
the pharmacokinetic profile and
determine the bioavailability of pure
Astaxanthin with astaxanthin
nanoemulsion via the oral route using
rabbits and the HPLC (High-Performance
Liquid Chromatography) method.
1. Method
2.1. Tools
The tools used in this study were
laboratory glassware (Pyrex), oral
syringe, stopwatch, analytical balance
(Toledo Meter), 1 mL syringe (Onemed),
3 mL syringe (Onemed), heparin tube,
vortex (MixMat), centrifugation (PLC-
05), Eppendorf tube (Onemed), dropper,
micropipette, sonicator (power sonic420),
vial injector. HPLC (Waters e2695), UV
detector, XTerra® column Sunfire C18
5µm (4.6x150mm). Mobile phase
Methanol: water milliQ: ethyl acetate
(82:8:10(v/v)). With a flow rate of 1.2
mL/minute at a wavelength of 474 nm,
the retention time was 2.90-3.50 minutes,
and the sample was measured by the
isocratic method.
2.2 Materials
The materials used in this study
were standard Astaxanthin 97% (HPLC)
from Blakeslea trispora, pure
Astaxanthin, astaxanthin nanoemulsion,
Acetone (merck), Methanol for HPLC
grade (Sigma Aldrich), Water milliQ
L. Nurdianti et al / Indo J Pharm 5 (2023) 441-448
443
(merck), Ethyl Acetate for HPLC grade
(Merck), Male Rabbits with a body
weight of 1.5 - 2 kg, ethanol, and
tetrahydrofuran (Merck).
2.3. Selection of Test Animals and
Dosage
The test animals were healthy
male rabbits with a body weight of 1.5-2
kg. The number of rabbits used was 16,
divided into two groups. A single dose of
pure Astaxanthin and astaxanthin
nanoemulsion via the oral route was
given at 7 mg/1.5 kg BW Rabbit. Before
the administration of astaxanthin, the
blood sample of the rabbit was
determined to be negative control.
2.4. Plasma Preparation
5 mL of blood was taken from the ear
(marginal vein) using a syringe and then
put into a heparin tube. Then, it was
centrifuged for 20 minutes at 1,500 rpm,
and the plasma was taken. Plasma was
then transferred to an eppendorf tube and
stored in a refrigerator (-200C) (Meor et
al., 2012). Blood samples were taken
from the ear at intervals of about 0, 1, 2,
3, 4, 6, 8, 10, 12, 24, 48, 72, and 92 hours.
2.5. Preparation of standard solutions
and Astaxanthin concentration series
Astaxanthin was dissolved with acetone
p.a., and a calibration curve of standard
astaxanthin was made and added to rabbit
plasma with a concentration between
(0.25-10 mg/L).
3. Method Validation
3.1 Linearity
Linearity was evaluated from the 0.25 -
10 mg/L calibration curve. Drug-free
rabbit plasma was dissolved in a standard
solution of astaxanthin with a
concentration of 0.25, 0.75, 1.25, 2.5, 5,
8, and 10 mg/L. The calibration curve
was obtained by plotting the peak area of
astaxanthin with astaxanthin
concentration and then determining the
correlation coefficient (r).
3.2. Accuracy and Precision
Determine the accuracy using (%
recovery) by making three analyte
concentrations with the equation:
% Recovery = 
  (Eq.1)
L. Nurdianti et al / Indo J Pharm 5 (2023) 441-448
Information (Eq.1) :
CF = The total concentration of the sample obtained from the measurement
CA = Actual sample concentration
C*A = Concentration of added analyte
Determining Precision by using % RSD (Relative Standard Deviation) with the equation:
%RSD =
  (Eq. 2)
Information (Eq. 2) :
SD = Standard Deviation.
= The average level of Astaxanthin in the sample.
2.6.3 Determination of LOD and LOQ
The detection limit (LOD) and the quantification limit (LOQ) are calculated by the
following formula:
LOD = 3α / S
LOQ = 10α / S
444
3.3. Blood Sample Preparation
Plasma samples stored in a
refrigerator (-200C) before analysis were
thawed at room temperature. 200 µL of
plasma was taken and put into a
microcentrifugation eppendorf tube and
deproteinized using 200 µL of a mixture
of ethanol and tetrahydrofuran (1:9). The
mixture was vortexed for 2 min and then
centrifuged at 14,000 rpm for 20 min.
After being centrifuged, it was put into an
injector vial, taken by auto injection of as
much as 10 µL into the HPLC, and
measured the area under the peak of the
chromatogram (Meor et al., 2012).
3.4. Data Analysis
In this experiment, the analysis
will be used using Microsoft Excel 2010
to determine pharmacokinetics. Statistical
analysis of the AUC0-∞ comparison
between groups using One Way ANOVA
95% confidence. Statistical analysis was
performed using SPSS version 23.0.
4. Result
4.1. Analysis Method Validation
Results
4.1.1. Linearity
Astaxanthin standard curve
manufacture using rabbit plasma was
measured using HPLC at a wavelength of
474 nm, resulting in a calibration curve of
0.25-10 mg/L, which obtained r2 = 0.995.
The results of the standard astaxanthin
calibration curve can be seen in Figure 1.
L. Nurdianti et al / Indo J Pharm 5 (2023) 441-448
Figure 1. Astaxanthin Standard Calibration Curve Results (Plasma was extracted with
standard astaxanthin)
Figure 2. Chromatogram of Astaxanthin under the following conditions: mobile phase
Methanol: Water: Ethyl acetate (82: 8: 10 v/v), flow rate 1.2 mL/min, Isocratic method
445
Accuracy and Precision
The accuracy results show that the
replica value from the analysis results is
getting closer to the actual sample value
with a % Recovery of 102.93, 103.11, and
102.25. These results are still within the
range accepted by the FDA, which is 80-
120%. The results of precision
measurements obtained values of % RSD
1.33, 1.65, and 2. These results meet the
FDA criteria of not more than 15%.
3.1.2. Determination of LOD and LOQ
LOD and LOQ measurement results. The
value for LOD is 0.08 mg/L, and LOQ is
0.25 mg/L.
4.1.3. Astaxanthin Pharmacokinetic
Profile Results
The results of measuring
astaxanthin plasma concentrations from 0
hours to 96 hours on pure Astaxanthin
and astaxanthin nanoemulsions are
presented in Figure 3. Then, the result
parameters of the pharmacokinetic profile
of Astaxanthin can be seen in Table 1.
L. Nurdianti et al / Indo J Pharm 5 (2023) 441-448
-3
-2,5
-2
-1,5
-1
-0,5
0
0,5
1
1,5
020 40 60 80
Ln Concentration (mg/L)
Time (hours)
Pure Astaxanthin
Astaxanthin
Nanoemulsion
Figure 3. Astaxanthin Plasma Test Results
Table 1. Parameter Results from Pharmacokinetic Profile of Astaxanthin
Pharmacokinetic parameters Astaxanthin Astaxanthin
nanoemulsion
Ka0,0638±0,0072 0,9663±0,0199
Ke0,0309±0,0024 0,0480±0,0019
t1/2abs (hours) 10,95±1,241 0,71±0,0189
t1/2e (hours) 22,53±1,7492 14,50±0,6447
tmax (hours) 21,73±2,3619 3,27±0,0896
AUC0-∞ (mg.hours/L) 74,9127±13,2619 95,2615±2,1310
Vd (L) 2,496±0,2496 1,2273±0,0520
Cl (L/hours) 0,0763±0,0145 0,0588±0,0013
Cmax (mg/L) 1,1723±0,1023 3,9057±0,1585
5. Discussion
The results of the measurement of
astaxanthin plasma concentrations from 0
hours to 96 hours on pure Astaxanthin
and astaxanthin nanoemulsions are
presented in Figure 3 showing the plasma
concentration curve of pure Astaxanthin
and astaxanthin nanoemulsions showing
one-compartment model in the form of
monophase (absorption and elimination
phases), based on the research conducted
by ( Odeberg, Lignell, Pettersson, &
446
L. Nurdianti et al / Indo J Pharm 5 (2023) 441-448
Höglund, 2003) on Astaxanthin
formulated in lipid-based and (Maltby,
Albright, Kennedy, & Higgs, 2003) on
Astaxanthin dissolved in sesame oil and
gelatin.
Parameter testing of Astaxanthin
Pharmacokinetic profiles based on
calculations in Table 1 Parameters of
absorption speed (Ka) and elimination
speed (Ke) in two dosage forms of pure
astaxanthin showed faster changes in
astaxanthin nanoemulsions with ka values
of 0.9663-0.0480. In addition, pure
astaxanthin resulted in a t1/2 absorption
calculation 10.95 hours longer than
astaxanthin nanoemulsions.
The value of elimination half-life (t1/2e) is
inversely proportional to the speed of
elimination, so the lower the elimination
speed value, the higher the elimination
half-life (t1/2e) (Katzung, 2011), which
means that the time required for
astaxanthin to be eliminated is longer.
This is shown in pure astaxanthin, and the
t1/2 elimination calculation is 22.53 hours
longer than the astaxanthin
nanoemulsion, which is a 14.50-hour t1/2
elimination. This does not deviate from
the results of previous studies on the
pharmacokinetics of astaxanthin,
reporting elimination half-lives of 21
hours (Østerlie, Bjerkeng, & Liaaen-
Jensen, 2000) and 15.9 hours (Odeberg et
al., 2003).
The results showed that astaxanthin
nanoemulsions had the highest absorption
speed, as indicated by a smaller tmax value
of 3.27 hours, compared to pure
astaxanthin, which was about 21.73
hours. This is because a decrease in the
absorption rate indicates that the drug is
slowly absorbed by the body, causing a
decrease in tmax. If the tmax value is small
at peak times (tmax), Cmax is reached
quickly. However, if the peak time value
(tmax) is large, the peak level (Cmax) will
be reached for a long time (Shargel et al.,
2005).
Based on the calculation results, the peak
concentration (Cmax) is greater in the
Astaxanthin nanoemulsion, namely Cmax
3.9057 mg/L, compared to pure
Astaxanthin Cmax 1.1723 mg/L.
The clearance in the Astaxanthin
nanoemulsion was smaller,
0.0588±0.0013 L/hour, followed by pure
Astaxanthin at about 0.0763±0.0145
L/hour. The volume of distribution (Vd)
is smaller in the Astaxanthin
nanoemulsion at 1.2273 L compared to
pure Astaxanthin. The relatively high
volume of distribution indicates
considerable absorption, binding, or
metabolism of astaxanthin by
extravascular tissues (Østerlie et al.,
2000). Statistically, the difference in
AUC0-∞ in pure Astaxanthin and
Astaxanthin nanoemulsion was
significant (P<0.05), so the average of
each group was significantly different in
the dosage form group—the average
results of AUC0-∞ in Table 1. The
Astaxanthin nanoemulsion is larger,
namely 95.2615±2.1310 mg.hour/L,
followed by pure Astaxanthin at around
42.8276±0.8739 mg. hour/L. Astaxanthin
nanoemulsion showed much greater drug
absorption according to research (Meor
Mohd Affandi, Julianto, & Majeed, 2012)
on Astaxanthin formulated in nanosized
and research (Domínguez-Hernández,
García-Alvarado, García-Galindo,
Salgado-Cervantes, & Beristáin, 2016) on
Astaxanthin formulated in the form of
nanoemulsions showed that Self-
Nanoemulsions increased drug absorption
much greater when surfactants were
combined with lipophilic and hydrophilic
added in the formulation, thereby
increasing the surface area of the drug
447
L. Nurdianti et al / Indo J Pharm 5 (2023) 441-448
and increasing its solubility and
permeation.
6. Conclusion
Pharmacokinetic Profile
Astaxanthin has a significant difference in
AUC0-∞ between Astaxanthin
nanoemulsion and pure astaxanthin
administered orally. Increasing the
AUC0-∞ value indicates a much greater
drug absorption, affecting the increase in
the bioavailability of astaxanthin. The
effect of astaxanthin formulation in
nanoemulsion can increase the solubility,
dissolution, and absorption of lipophilic
drugs.
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