Journal of Chromatography B, 972 (2014) 95–101

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Development and validation of a rapid HPLC method for the

quantification of GSE4 peptide in biodegradable PEI–PLGA
nanoparticles
Susana P. Egusquiaguirre a,b , Cristina Manguán-García c , Rosario Perona c ,
José Luís Pedraz a,b , Rosa Maria Hernández a,b , Manuela Igartua a,b,∗
a
NanoBioCel Group, Laboratory of Pharmaceutics, University of the Basque Country, Faculty of Pharmacy, Paseo de la Universidad, 7, 01006
Vitoria-Gasteiz, Spain
b
Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Paseo de la Universidad, 7, 01006
Vitoria-Gasteiz, Spain
c
Instituto de Investigaciones Biomédicas CSIC/UAM, IDIPaz and CIBER de Enfermedades Raras CIBERER, Arturo Duperier, 4, 28029 Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history: In this work a high performance liquid chromatographic (HPLC) method has been developed and validated
Received 8 July 2014 for the content determination of GSE4 peptide in PEI–PLGA nanoparticles. Chromatographic separation
Accepted 27 September 2014 was performed on a C18 column, and a gradient elution with a mobile phase composed of methanol
Available online 5 October 2014
and 0.1% aqueous trifluoroacetic acid (TFA) solution, at a flow rate of 1 ml/min, was used. GSE4 peptide
identification was made by fluorescence detection at 290 nm. The elution of methanol:TFA was initially
Keywords:
maintained at (20:80, v/v) for one min and the gradient changed to (80:20, v/v) in 6 min. This ratio
GSE4
was then followed by isocratic elution at (80:20, v/v) during another min and for further 3 min it was
HPLC
Nanoparticles
linearly modified to (20:80, v/v). The developed method was validated according to the ICH guidelines,
PEI being specific, linear in the range 10–100 ␮g/ml (R2 = 0.9996), precise, exhibiting good inter-day and
PLGA intra-day precision reflected by the relative standard deviation values (less than 3.88%), accurate, with a
Validation recovery rate of 100.18 ± 0.95%, and stable for 48 h at 5 ◦ C or at RT when encapsulated in nanoparticles.
The method was simple, fast, and successfully used to determine the peptide content in GSE4-loaded
PEI–PLGA nanoparticles.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction sequence of a component of telomerase (dyskerin), has been sug-

Syndromes of telomere maintenance are highly heterogeneous
rare inherited disorders characterized for defects on telomere
elongation. These syndromes are all premature aging diseases asso-
ciated with shortened telomeres, caused by mutations in any of
the components of the telomerase complex, or in components
associated with telomerase, and presenting an increased predispo-
sition to cancer [1]. Unfortunately, there is not a specific treatment
for any of these disorders, just medication to palliate the symp-
toms. Recently, the peptide GSE24.2, corresponding to an internal
∗ Corresponding author at: NanoBioCel Group, Laboratory of Pharmaceutics, Uni-
versity of the Basque Country, Faculty of Pharmacy, Paseo de la Universidad, 7, 01006
Vitoria-Gasteiz, Spain. Tel.: +34 945 01 3875; fax: +34 945 013040.
E-mail addresses: susanapatricia.egusquiaguirre@ehu.es (S.P. Egusquiaguirre),
cmanguan@iib.uam.es (C. Manguán-García), rperona@iib.uam.es (R. Perona),
joseluis.pedraz@ehu.es (J.L. Pedraz), rosa.hernandez@ehu.es (R.M. Hernández),
manoli.igartua@ehu.es (M. Igartua).
gested as a promising approach for the treatment of syndromes
of telomere shortening due to the capacity of this peptide to res-
cue cells with defective telomerase from premature senescence,
reactivating the deficient telomerase [2]. However, despite its great
therapeutic value, the clinical application of bioactive peptides is
limited by their poor stability, short circulation half-life and low
permeability across barriers [3,4]. For this reason, their entrap-
ment into micro- or nanoparticles has the potential to enhance
their therapeutic activity, avoiding the mentioned limitations
[4,5]. In a previous study, the peptide GSE24.2 was encapsu-
lated into poly(d,l-lactic-co-glycolic acid) (PLGA) and PLGA–PEG
nanoparticles, uncoated or coated with polyethyleneimine (PEI)
as a polycation, or with different cell penetrating peptides (CPPs),
with an improved cellular uptake and telomerase reactivation.
Therefore, these formulations were proposed as promising candi-
dates for the intracellular delivery of GSE24.2 to treat Dyskeratosis
Congenita (DC) and other defective telomere disorders (Pat. No.
P201330131). However, GSE24.2 peptide is biologically obtained

http://dx.doi.org/10.1016/j.jchromb.2014.09.041
1570-0232/© 2014 Elsevier B.V. All rights reserved.
96 S.P. Egusquiaguirre et al. / J. Chromatogr. B 972 (2014) 95–101
from Escherichia coli, with a laborious, costly, and long obtaining
process; so a synthetic peptide easier and faster to obtain was
needed. Accordingly, in this paper we present the synthetic pep-
tide GSE4, corresponding to the amino acidic therapeutic sequence
of GSE24.2 (GFINLDKPSNP) but chemically obtained by the com-
pany China Peptides Co. Ltd. Thereby, since cationic PEI–PLGA
nanoparticles have been proved to enhance the internalization of
such nanoparticles in cells, and increase the therapeutic benefits of
GSE24.2 peptide, in this work we have encapsulated the synthetic
peptide GSE4 into PEI–PLGA nanoparticles.
Once GSE4-loaded nanoparticles were prepared, we tried to
quantify the content of encapsulated GSE4 peptide through col-
orimetric techniques, using the Lowry assay (by the micro-BCA
technique [6]), the same way the quantification of GSE24.2
encapsulated was estimated. Therefore, in this paper, PEI–PLGA
nanoparticles containing GSE4 peptide were prepared, and hence,
to fully characterize these nanoparticles, the main objective of this
work was to develop and validate a specific and simple High Per-
formance Cromatographic (HPLC) method for the quantification of
GSE4 content in PEI–PLGA nanoparticles.
2. Experimental
2.1. Reagents and chemicals
The materials used for the elaboration of the nanoparticles
were the following: the polymer poly(d,l-lactide-co-glycolide)
PLGA (Resomer RG® 503) 50:50 (lactic/glycolic %), with a molec-
ular weight of 33.9 kDa and an intrinsic viscosity of 0.32–0.4 dl/g
was purchased from Boehringer Ingelheim (Ingelheim, Germany).
Dichloromethane (DCM) HPLC grade as the organic solvent, 2-
propanol and poly-ethylene-glycol 400 (PEG 400) were purchased
from Panreac (Barcelona, Spain). Sodium alginate MVG [Mw
200,000–300,000 g/mol, medium viscosity, monomer ratio 60:40
guluronic/mannuronic (%)] was supplied by Pronova UP, Nova-
Matrix FMC BioPolymer (Sandvika, Norway). The surfactant used
in the emulsification process was poly(vinyl alcohol) (PVA, with
an average MW of 30–70 kDa, 87–90% hydrolysis degree), as
well as polyethyleneimine (PEI with a MW of 25 kDa, branched),
calcium chloride (CaCl2 ·2H2 O with a MW of 147.01 g/mol), and
d-trehalose (TRH with a MW of 378.33 g/mol) were obtained
from Sigma–Aldrich (Barcelona, Spain). GSE4 peptide was supplied
by China Peptides Co. Ltd. The materials used for the develop-
ment of the analytical method were the following: HPLC-grade
methanol was purchased from Scharlab (Sentmenat, Spain), tri-
fluoroacetic acid (TFA) UVASOL® was acquired from Merck KGaA
(Damstadt, Germany) and dimethyl sulphoxide (DMSO) from Pan-
reac (Barcelona, Spain). Water was purified using a Milli-Q Plus
system (Millipore) with conductivity of 18 M. Mobile phase sol-
vents were degassed in an ultrasonic bath for 15 min before use.
2.2. Preparation of GSE4-loaded PEI–PLGA nanoparticles
The GSE4-loaded nanoparticles were obtained by a double-
emulsion solvent evaporation technique. Briefly, PLGA was
dissolved in dichloromethane with 1.3% (w/w) polyethyleneimine
(PEI) as a polycation, and an aqueous solution of 2% (v/w) GSE4
in 1% (w/v) alginate (ALG) with 2.5% (v/v) polyethyleneglycol 400
(PEG 400) was added to the organic solution while sonicating dur-
ing 30 s with ultrasounds using a Sonicator (Branson Ultrasonic
Sonifier® 250) in an ice bath, to form an oil-in-water emulsion. This
first emulsion was rapidly poured into a 5% (w/v) polyvinyl alcohol
(PVA) aqueous solution with 5% (w/v) CaCl2 , to allow the gela-
tion of ALG, and emulsified by means of sonication for 60 s which
resulted in a double emulsion (W/O/W). Next, the double emulsion
was incorporated into a 2% (w/w) alcoholic aqueous solution of iso-
propanol (IPA) with 5% (w/v) CaCl2 in order to favor the evaporation
of the organic solvent by stirring the system during 2 h. The parti-
cles were then recovered by ultracentrifugation (25,000 × g, 15 min,
4 ◦ C, Sigma 3–30 K) and washed three times with water to remove
the surfactant. The nanoparticles were dispersed with the cryopro-
tectant trehalose (15%, w/w), and the resulting nanosuspension
was subsequently cooled to −18 ◦ C and freeze-dried (LyoBeta 15,
Telstar, Tarrasa, Spain).
2.3. Equipment
The HPLC system used to perform all chromatographic runs
consisted of a Waters 2795 Alliance (Milford, MA, USA) com-
bined with a multi ␭ fluorescent detector (Waters 2475), and
was equipped with a vacuum degasser, a quaternary pump, an
autosampler, and an automatic injector. Empower 3 chromatog-
raphy software (Milford, MA, USA) was used for the equipment
control, data acquisition, analysis and reporting. The HPLC anal-
ysis was conducted by using a C18 column (Thermo Scientific BDS
Hypersil, Phenomenex, Torrance, CA, USA) with a 5 ␮m particle size,
4.6 mm internal diameter, and 100 mm length, maintained at room
temperature.
Mass spectrometric analysis was carried out using a micromass
ZQ (Waters) mass spectrometer equipped with an electron spray
ion (ESI) source. Instrument control and data acquisition were per-
formed using software MassLynxTM v 4.0 (Waters).
2.4. Chromatographic conditions
Chromatographic analyses were performed in the gradient
mode. The mobile phase consisted of methanol as solvent A and
0.1% trifluoroacetic (TFA) aqueous solution as solvent B, initially set
in the ratio (20:80, v/v) for one min, and linearly changed to (80:20,
v/v) over 6 min. This ratio was kept for another min and from 7 to
10 min it was linearly modified to its original rate, (20:80, v/v) in
order to re-equilibrate the column prior to the following injection.
The total run time was 10 min. Eluent was pumped at a flow rate of
1 ml/min, with a sample injection volume of 50 ␮L and a detection
wavelength at 290 nm. All experiments were performed at room
temperature (RT) and the total area of peak was used to quantify
GSE4.
2.5. Preparation of standard and sample solutions
A freshly prepared GSE4 stock standard solution of 1 mg/ml was
prepared by dissolving GSE4 peptide in purified water, and subse-
quent dilutions were carried out to obtain five standard solutions
at different concentrations over the range of interest (10, 25, 50, 75,
and 100 ␮g/ml). These solutions were prepared daily prior to the
injection into the system.
In the case of GSE4-loaded nanoparticles test solutions, the
proposed analytical procedure is based on the breakage of the
nanoparticles by dissolution of the polymer in an organic solvent
and the subsequent extraction of the peptide in an aqueous phase.
For this purpose, 50 ␮l of DMSO were added to approximately 5 mg,
exactly weighted, of freeze-dried GSE4-loaded PEI–PLGA nanopar-
ticles. The mixture was vigorously stirred in a vortex stirrer until the
complete breaking of the nanoparticles and then it was dissolved in
950 ␮l of purified water. Samples were vigorously stirred for 10 min
in order to extract the peptide from the organic solvent to the
aqueous phase, and then the aqueous phase was separated by ultra-
centrifugation (25,000 × g, 10 min, 4 ◦ C, Sigma 3–30 K). Finally, the
peptide concentration was analyzed by HPLC and it was determined
with a previously constructed analytical curve. These analyses were
performed in triplicate.
 S.P. Egusquiaguirre et al. / J. Chromatogr. B 972 (2014) 95–101
2.6. Method validation
The objective of validating an analytical procedure is to demon-
strate that it is adequate for its intended purpose. The HPLC method
was validated in agreement with the International Conference
on Harmonization (ICH) guidelines [7] in terms of the following
analytical parameters: specificity, linearity, precision (intra- and
inter-day), and accuracy. The stability of GSE4 standard solutions
and GSE4-loaded nanoparticles test solutions was also studied.
2.6.1. Specificity
The specificity was performed in order to determine the abil-
ity of the analytical method to exactly measure the concentration
of GSE4 loaded into nanoparticles without interferences due to
constituents that may be present. In this assay, specificity was
evaluated by comparison of the peak area corresponding to
GSE4-loaded nanoparticles test solution with the representative
chromatograms of samples containing possible interfering sub-
stances (i.e. blank nanoparticles and solutions from each of the
components of the mobile phase).
2.6.2. Linearity
The linearity study verifies that the sample solutions are in a
concentration range where analyte response is linearly propor-
tional to the concentration. Linearity was established by calculation
of a regression line from the graphical plot of the chromatographic
peak area (y) versus GSE4 concentration (x) of five standard solu-
tions (10, 25, 50, 75, and 100 ␮g/ml), obtaining a calibration curve
and performing the corresponding statistical study using a lin-
ear least-squares regression methodology and by analysis of the
respective response factors (i.e. peak area divided by concentration
of each standard sample). Each level of concentration was prepared
in triplicate. Besides, by means of analysis of variance (ANOVA) and
‘t’ Student’s test, the validity of the method was verified.
2.6.3. Precision
The precision is the parameter that indicates the closeness of
agreement between a series of measurements obtained from mul-
tiple analysis of the same sample under the established conditions.
In our study, the precision was assessed at three different levels:
instrumental precision and method repeatability for intra-day pre-
cision, and intermediate precision for inter-day precision.
2.6.3.1. Instrumental precision. The instrumental precision eval-
uated the repeatability of the instrumental system and it was
tested by performing six replicate injections of a standard solution
(50 ␮g/ml) from the same vial and in the same day. The standard
deviation (SD) and the relative standard deviation (RSD) were cal-
culated for the six injections. For acceptance, the RSD value had to
be smaller or equal to 1.37%.
2.6.3.2. Method repeatability. The method repeatability indicates
the precision under the same operating conditions over a short
interval of time. The method repeatability of the measurements
was assessed by the analysis of six independent standard solu-
tions (50 ␮g/ml), and six independent GSE4-loaded nanoparticles
test solutions injected in triplicate in the same day. The SD and
the RSD were calculated for the six measurements. The method
repeatability can be accepted when RSD is smaller or equal to 1.94%.
2.6.3.3. Intermediate precision. The intermediate precision ana-
lyzes the effect that random events have on the method
performance. The intermediate precision was evaluated by ana-
lyzing six standard solutions (50 ␮g/ml) and six GSE4-loaded
nanoparticle test solutions, injected in triplicate, on two different
days and by two different analysts. The results were reported as the
97
Table 1
System suitability parameters. The results are expressed as mean value ± SD.
Chromatographic parameters Result Acceptance criteria
GSE4 retention time (min) 5.56 ± 0.01 –
Retention factor, k 4.56 ± 0.01 2 < k < 10
Tailing factor, T 1.32 ± 0.06 0.8–1.5
Number of theoretical plates, N 11,847 ± 155 N > 2000
SD and the RSD The method repeatability can be accepted when RSD
is smaller or equal to 3.88%.
2.6.4. Accuracy
According to ICH guidelines [7] and USP 36 NF 31 [8], the accu-
racy of an analytical method expresses the proximity between
obtained experimental results and the real results of the sample.
The accuracy was evaluated by comparing the theoretical value
with the percentage recoveries of the mean concentration of GSE4
at three different concentration levels (lower level 25%; middle
level 50% and higher level 100%) precisely prepared, where known
concentrations of GSE4 standard solutions were prepared (25, 50
and 100 ␮g/ml). The mean concentration value obtained for each
level was compared to the theoretical value, which was considered
to be 100%, and the RSD was determined.
2.6.5. Stability
Tests were also performed to determine the stability of GSE4 in
the standard solution (50 ␮g/ml) or extracted from nanoparticles.
Stability was evaluated at two different thermal conditions: room
temperature (RT) and 5 ◦ C, quantifying the concentration of GSE4
over a period of 8 h, 24 h and 48 h. Standard and test samples were
considered stable if the GSE4 mean concentration was smaller or
equal to than 95%. Tests were performed in triplicate.
3. Results and discussion
3.1. Optimization of the chromatographic method
The proposed method was developed with the purpose to
provide a simple and optimized HPLC method for the assessment
of GSE4 encapsulated in PEI–PLGA nanoparticles. The chromato-
graphic conditions and performance parameters were adjusted to
provide a simple analysis with the best peak resolution, reducing
run time and lowering the cost of analysis. The procedure was ini-
tially based on the existing method developed by the company
which synthesized the GSE4 peptide, using a solvent A composed of
acetonitrile (ACN) and a solvent B composed of 0.1% TFA in aqueous
solution, as a mobile phase, and a determined gradient. However,
as the concentrations that we were working with were much lower
than those used during the synthesis, the use of this method in our
case resulted in asymmetric peaks and very difficult to detect. In
order to overcome these problems, we decided to vary the gradi-
ent of the solvents, and hence, the peak of the peptide obtained
after this variation was detected, but overlapped with the signal
generated by the mobile phase. Then we tried to solve this issue
by changing the organic solvent to methanol and modifying the
gradient of the mobile phase. With this method, there was a sub-
stantial separation of the signal of the mobile phase (approximately
around 7 to 8.6 min) and the peak of the peptide (at 5.5 min), so
therefore, the final method conditions, described in materials and
methods, were established. The resulting suitability parameters of
the chromatographic setup for validation are presented in Table 1.
98 S.P. Egusquiaguirre et al. / J. Chromatogr. B 972 (2014) 95–101
Fig. 1. Representative chromatograms corresponding to a standard solution of GSE4 (100 ␮g/ml) (dotted-dashed line), GSE4-loaded PEI–PLGA nanoparticles (dashed line),
blank PEI–PLGA nanoparticles (solid line), and mobile phase signal (dotted line).
3.2. Method validation
3.2.1. Specificity
In order to evaluate the specificity of the method, the chro-
matograms of GSE4 and those of potential interfering formulation
components were compared. Blank nanoparticles solution, and
solutions from each of the components of the mobile phase
(i.e. methanol, purified water, and 0.1% (w/w) TFA) were ana-
lyzed according to the proposed method and compared with the
chromatogram of GSE4 standard samples. Overall, obtained data
provide evidence that the method can be regarded as specific
since the chromatograms show an absence of any peak in the
region where GSE4 is eluted. Therefore, our results verify the
selectivity of the proposed method. Fig. 1 shows the representa-
tive chromatograms of GSE4 standard solution (100 ␮g/ml) and
PEI–PLGA nanoparticles containing GSE4, both with a retention
time of 5.5 min, showing in both cases no interfering peaks. Addi-
tionally, in Fig. 1 we can also appreciate the chromatograms of
blank PEI–PLGA nanoparticles and the mobile phase, with no other
peaks corresponding to any of the components of the nanoparticle
formulation or solvent.
Moreover, in order to confirm the results obtained with
fluorescence detection, regarding GSE4 peptide identification,
HPLC–ESI-MS was employed. GSE4 peptide extracted from
PEI–PLGA nanoparticles was successfully identified by analyzing
the full scan mass spectra of the chromatographic peak obtained
at the same retention time as GSE4 standard solution. As shown in
Fig. 2, the masses of the peptide extracted from nanoparticles test
solution (A) and GSE4 standard solution (B) were identical.
3.2.2. Linearity
Linearity was studied by calculating the regression equation and
the correlation coefficient (R2 ) of a calibration curve plotting for five
standard solutions of different concentrations ranging from 10 to
100 ␮g/ml by the method of least squares. The calibration curve
and correlation coefficient were:
Area = 63, 471.33(±0.198)C + 92, 395.667(±3.326),
with a R2 = 0.9996
The standard deviation values are indicated in brackets. The R2
is higher than 0.999, indicating a good linearity in the established
range [9,10]. The suitable range for GSE4 analyses from 10 to
100 ␮g/ml was also confirmed by the response factor versus GSE4
standard solution concentration, with a linear curve slope close
to zero (slope = 0.023) indicating a linear response obtained over
the specified concentration range. According to the statistical anal-
ysis by ANOVA and ‘t’ Student’s test, the analytical method was
demonstrated to be linear, with a p < 0.05 (Table 2) [11,12].
3.2.3. Precision
In the case of the developed method, precision tests accom-
plished at three different levels were: instrumental precision (n = 6)
and method repeatability (n = 6), corresponding to the intra-day
precision, and intermediate precision (n = 12), for the inter-day
precision. These precision analyses were expressed by the cor-
responding RSD values. Table 3 summarizes all the precision
results of the GSE4 standard solutions (50 ␮g/ml) and GSE4-loaded
nanoparticles test solutions (the concentration varied according to
the encapsulation efficiency), respectively. For instrumental pre-
cision and method repeatability, the RSD values of GSE4 standard
solutions were 0.35 and 1.06, respectively; and as for the inter-
mediate precision, the RSD was 1.71. In the case of GSE4-loaded
nanoparticles test solutions, instrumental precision was 0.27 and
intermediate precision 1.31. All results were below the established
limit according to the variation accepted (RSD ≤1.37%, ≤1.94% and
≤3.88% for the instrumental precision, method repeatability and
intermediate precision, respectively), indicating a low variability
between the values obtained, and a satisfactory precision of the
analytical method [13].
3.2.4. Accuracy
Accuracy of the analytical method or recovery was assessed by
calculating the percentage recoveries of a known amount of the
analyte at three different concentrations. The percentage recov-
ery was determined by comparing the mean concentration value
obtained for each level with the theoretical value, which was con-
sidered to be 100%. Three standard solutions (10, 50 and 100 ␮g/ml)
were carefully prepared in triplicate as described above and ana-
lyzed by the proposed method, obtaining recovery values around
99–100% (99.49 ± 1.01%, 100.13 ± 1.01 and 100.92 ± 0.17% for 10,
50 and 100 ␮g/ml GSE4 standard solution, respectively). The over-
all mean recovery was found to be 100.18 ± 0.95% (n = 9), showing
strong agreement between experimental and theoretical values,
 S.P. Egusquiaguirre et al. / J. Chromatogr. B 972 (2014) 95–101 99

Fig. 2. Full scan mass spectra of GSE4 extracted from PEI–PLGA nanoparticles (A) and GSE4 standard solution (B).

and thus, it can be emphasized a suitable accuracy for the analytical
method.
3.2.5. Stability
Stability tests were carried out at 8 h, 24 h and 48 h, storing GSE4
standard solutions and GSE4-loaded nanoparticles test solutions
at RT and 5 ◦ C. The evaluation of the stability was based on the
recovery values of the peptide, and was determined by compar-
ing the mean concentration value obtained for each time with the
concentration value at 0 h for GSE4 standard solution and GSE4
extracted from nanoparticles, which was considered to be 100%.
Obtained results are presented in Table 4, showing that, when GSE4
standard sample was stored at RT, the peptide was stable till 24 h
(96.01%), but we could appreciate a slight decrease of the concen-
tration after 48 h (93.43%). When the standard sample was kept at
5 ◦ C, the peptide remained stable for at least 48 h, exhibiting values
over 95% for all the time points (95.87% was recovered at 48 h). In
the case of GSE4 extracted from nanoparticles, when it was kept
100 S.P. Egusquiaguirre et al. / J. Chromatogr. B 972 (2014) 95–101

Table 2
Statistical analysis of linearity.

Parameter t-Statistic p-Value Lower 95% Upper 95%

Multiple regression analysis

Intercept 1.940 0.044 −4255.651 79,289.027
Slope 203.804 0.000 63,500.171 64,860.826

Parameter Sum of squares Degrees of freedom Mean of squares F p-Value

ANOVA
Regression 6.586 1 6.586 41,536.096 0.000
Residual 2.061 13 1.586
Total 6.589 14

Table 3
Summary of precision results for GSE4 standard solutions and GSE4-loaded PEI–PLGA nanoparticles.

Mean measured SD (%) RSD (%)

concentration (␮g/ml)

Standard solution (␮g/ml)

Instrumental precision (n = 6) 49.14 0.17 0.35
Method repeatability (n = 6) 49.86 0.53 1.06
Intermediate precision (n = 12) 49.17 0.84 1.71

Nanoparticles test solution (␮g/ml)

Method repeatability (n = 6) 34.77 0.09 0.27
Intermediate precision (n = 12) 34.73 0.45 1.31

Table 4 Peptide loading, represented as the ␮g of GSE4 per mg freeze-dried

Stability results of GSE4 standard solution and GSE4-loaded PEI–PLGA nanoparticles
nanoparticles, was calculated by dividing the amount (␮g GSE4/ml)
sample solution.
by 5 mg of nanoparticles, which was the amount of nanoparticles
Amount (␮g/ml) Mean percentage of initial dissolved in 1 ml of the dissolution media. The peptide loading was
concentration (%)
determined to be 6.20 ± 0.78 ␮g of GSE4 per mg of nanoparticles.
Standard solution (50 g/ml) Triplicate measurements were performed for each sample.
RT
0h 49.81 100
8h 48.61 97.59 4. Conclusions
24 h 47.82 96.01
48 h 46.54 93.43
In the present study we have developed and validated a
5 ◦C chromatographic method for the determination of GSE4 peptide
0h 49.81 100
encapsulated into PEI–PLGA nanoparticles according to the Vali-
8h 48.65 97.66
24 h 47.97 96.30
dation of Analytical Procedures ICH guideline. Obtained results
48 h 47.75 95.87 demonstrated that all parameters were within the limits proposed
by this guideline, indicating that this method is specific, linear,
Nanoparticles sample solution precise, and accurate within the range of 10–100 ␮g/ml. Further-
RT more, according to the stability results, GSE4 was stable for at least
0h 33.37 100
8h 32.62 97.74
48 h stored at 5 ◦ C either as a standard solution or extracted from
24 h 31.88 95.51 nanoparticles, with peptide recovery values above 95%. However,
48 h 30.91 92.62 when GSE4 was maintained at RT, it started to degrade after 24 h
5 ◦C
of storage in standard solutions or extracted from nanoparticles.
0h 33.37 100 Additionally, the present HPLC method can be considered simple,
8h 32.66 97.87 fast, and easy to apply, making it very suitable for routine analysis in
24 h 32.15 96.34 quality control of GSE4 peptide formulated in biodegradable PLGA
48 h 32.09 96.17
nanoparticles, being sample preparation and analytical procedure
run times short.
at RT, after 24 h a slight degradation could be appreciated, with a
recovery value of 92.62%, however the peptide remained stable for Acknowledgements
at least 48 h when maintained at 5 ◦ C, with recovery values over 95%
at each time point (Table 4). Therefore, according to these results, S.P. Egusquiaguirre thanks the Basque Government (Depar-
GSE4 peptide remained stable for at least 48 h either in standard tamento de Educación, Universidades e Investigación) for the
solution or extracted from PEI–PLGA nanoparticles at 5 ◦ C, while, fellowship research grant. We also acknowledge financial support
when it was stored at RT, after 48 h it started to slowly degrade. from the Spanish Government (Ministerio de Economia y Compet-
itividad) for the Subprogram INNPACTO (IPT-2012-0674-090000)
3.3. Method applicability and FEDER funds.

The proposed method was applied to study the quantification

References
of GSE4 peptide encapsulated into PEI–PLGA nanoparticles. The
developed method was confirmed to be effective, fast and meets [1] C.M. Kong, X.W. Lee, X. Wang, FEBS J. 280 (2013) 3180.
the criteria for method validation. These nanoparticles presented a [2] R. Machado-Pinilla, I. Sanchez-Perez, J.R. Murguia, L. Sastre, R. Perona, Blood
mean diameter of around 200 nm and a zeta potential of +24 mV. 111 (2008) 2606.
 S.P. Egusquiaguirre et al. / J. Chromatogr. B 972 (2014) 95–101 101

[3] C. Pinto Reis, R.J. Neufeld, A.J. Ribeiro, F. Veiga, Nanomedicine 2 (2006)
53.
[4] S.C. Yadav, A. Kumari, R. Yadav, Peptides 32 (2011) 173.
[5] A. Kumari, S.K. Yadav, S.C. Yadav, B. Colloids Surf, Biointerfaces 75 (2010) 1.
[6] K.J. Wiechelman, R.D. Braun, J.D. Fitzpatrick, Anal. Biochem. 175 (1988)231.
[7] International Conference on Harmonisation (ICH), Validation of Analytical Pro-
cedures: Text and Methodology, ICH Harmonised Tripartite Guideline, ICH
Topic Q2 (R1), 2005.
[8] USP 36 NF 31. The United States Pharmacopeia Convention, Rockville, MD.
[9] G.A. Shabir, J. Chromatogr. A 987 (2003) 57.
[10] N. Épshtein, Pharm. Chem. J. 38 (2004) 212.
[11] J.M. Sonnergaard, Int. J. Pharm. 321 (2006) 12.
[12] G.A. Shabir, J.W. Lough, S.A. Arain, T.K. Bradshaw, J. Liq. Chromatogr. Relat.
Technol. 30 (2007) 311.
[13] F. Bressolle, M. Bromet-Petit, M. Audran, J. Chromatogr. B: Biomed. Appl. 686
(1996) 3.