Method development for acetyl octapeptide-3 analysis by liquid chromatography-tandem mass spectrometry

Abstract

Background
Acetyl octapeptide-3 (SNAP-8) is an antiaging peptide that is more effective than acetyl hexapeptide-3, which is more stable than botulinum toxin and effectively relieves facial wrinkles. Products containing SNAP-8 such as patch are producing, but analytical method has not been reported to determination of SNAP-8.

Method
Mobile phase, collision energy, and desolvation line temperature were optimized, and mass spectral data set for SNAP-8 was newly constructed by liquid chromatography-triple quadrupole mass spectrometer in multiple reaction monitoring mode.

Results
The developed method showed good linearity ( r ≥ 0.9971) with limit of quantification of 0.0125 ng/mL, repeatability (% relative standard deviation = 0.02 to 0.12) and accuracy (% relative error = − 1.68 to 1.44) under optimal conditions. This method was successfully applied to a biodegradable microneedle patch loaded with SNAP-8.

Conclusion
The present method for the quantification of SNAP-8 may be useful for quality control in the cosmetic fields.

Full text

S H O R T R E P O R T Open Access
Method development for acetyl
octapeptide-3 analysis by liquid
chromatography-tandem mass
spectrometry
Moongi Ji
1†
, Hyeon-Seong Lee
1†
, Youngbae Kim
1
, Chan Seo
2
, Subin Choi
3
, Songjin Oh
1
, Jeuk Min
1
,
Hyung-Jin Park
1
, Jung Dong Kim
4
, Do Hyeon Jeong
4
and Man-Jeong Paik
1*
Abstract
Background: Acetyl octapeptide-3 (SNAP-8) is an antiaging peptide that is more effective than acetyl hexapeptide-
3, which is more stable than botulinum toxin and effectively relieves facial wrinkles. Products containing SNAP-8
such as patch are producing, but analytical method has not been reported to determination of SNAP-8.
Method: Mobile phase, collision energy, and desolvation line temperature were optimized, and mass spectral data
set for SNAP-8 was newly constructed by liquid chromatography-triple quadrupole mass spectrometer in multiple
reaction monitoring mode.
Results: The developed method showed good linearity (r≥0.9971) with limit of quantification of 0.0125 ng/mL,
repeatability (% relative standard deviation = 0.02 to 0.12) and accuracy (% relative error = −1.68 to 1.44) under
optimal conditions. This method was successfully applied to a biodegradable microneedle patch loaded with SNAP-8.
Conclusion: The present method for the quantification of SNAP-8 may be useful for quality control in the cosmetic fields.
Keywords: Acetyl octapeptide-3, Microneedle Patch, Multiple reaction monitoring mode, Liquid chromatography-tandem
mass spectrometry
Introduction
The synaptosome-associated protein of 25 kDa (SNAP-
25) is known to relate to facial wrinkles including
muscle contraction (Adler et al. 2001). The acetyl
hexapeptide-3 is a peptide fragment of SNAP-25 and it
has been widely used for the cosmetic materials of the
improvement of facial wrinkles because acetyl hexapep-
tide has an inhibiting effect on the function of SNAP-25
(Lipotec™). An acetyl octapeptide-3 (SNAP-8) added a
two-amino acid chain on acetyl hexapeptide-3, which
was designed for better effect of wrinkle improvement
than acetyl hexapeptide-3 (Lipotec™). A botulinum
toxin (BOTOX®) has been widely used for the im-
provement of facial wrinkles, but SNAP-8 has higher
safety than BOTOX® (Blanes-Mira et al. 2002;Zhou
et al. 2011). Both SNAP-8 and a botulinum toxin
(BOTOX®) target the SNAP-25. BOTOX® breaks the
SNAP-25 and causes muscle paralysis, while SNAP-8
captures the SNAP-25 and causes muscle relaxation
(Blasi et al. 1993; Gutiérrez et al. 1995;Gutierrez
et al. 1997; Blanes-Mira et al. 2002;Yamauchiand
Lowe 2004). Thus, SNAP-8 is more stable than
BOTOX®, which has been proposed as a functional
cosmetic material that can replace BOTOX® (Lipotec™;
Blanes-Mira et al. 2002). Although SNAP-8 is being
used in functional cosmetics, method for SNAP-8
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
* Correspondence: paik815@sunchon.ac.kr
†
Moongi Ji and Hyeon-Seong Lee contributed equally to this work.
1
College of Pharmacy, Sunchon National University, Suncheon 540-950,
Republic of Korea
Full list of author information is available at the end of the article
Journal of Analytical Science
and Technolog
y
Ji et al. Journal of Analytical Science and Technology (2020) 11:34
https://doi.org/10.1186/s40543-020-00232-8

analysis has not been reported. Patches are generally
used as an example of skin delivery products. Poly-
meric compounds such as peptides cannot pass
through the stratum corneum and skin absorption is
also difficult (Park et al. 2005). However, microneedle
patches penetrate the dermal layer and can transfer
substances such as SNAP-8 with large molecular
weights directly into skin, which were used for the
transdermal delivery of polymeric compounds (Zhang
et al. 2014). In this study, analytical method of
SNAP-8 was developed by liquid chromatography-
tandem mass spectrometry (LC-MS/MS). This method
was successfully applied to quantification of SNAP-8
level in biodegradable microneedle patches.
Materials and methods
Materials
SNAP-8 and the acetyl hexapeptide-3 as internal standard
(IS) were purchased from Lipotec (Barcelona, Spain); their
chemical structures are shown in Fig. 1.Biodegradable
microneedle patches containing SNAP-8 were provided
by Raphas Co. Ltd. (Seoul, Republic of Korea). High-
performance liquid chromatography (HPLC) grade water
and acetonitrile (ACN) were purchased from Daejung
Chemical (Siheung-si, Gyeonggi-do, Republic of Korea).
The syringe-driven filter unit (0.45 μm) was purchased
from Merck Millipore (Darmstadt, Germany). Formic acid
(FA) and trimethylamine (TEA) were purchased from
Wako Pure Chemical (Richmond, VA, USA). All other
chemicals were of analytical reagent grade.
Preparation of standard and calibration solutions
Stock solutions of SNAP-8 and IS were made up at
10.0 μg/μL in HPLC grade water. Working standard
solutions were prepared by diluting each stock solu-
tion to 12.5 ng/μL (SNAP-8) and 15.0 ng/μL(IS),
respectively. The SNAP-8 standards for quantitative
calibration curve were prepared from 0.0125 to 0.15
ng/μL by aliquots of each working standard solution.
All standard solutions were then stored at 4 °C.
Preparation of microneedle patches
Biodegradable microneedle of patch (1.42 mg) with
loaded SNAP-8 was extracted with distilled water (5 mL)
containing IS (150.0 ng). The extract was filtered with
syringe-driven filter unit (0.45 μm), then an aliquot of
10 μL was injected into the LC-MS/MS system.
Method validation for SNAP-8 analysis
Linearity, repeatability, accuracy, recovery, limit of
detection (LOD), and limit of quantification (LOQ)
were validated under optimal conditions with blank
patch matrix. Calibration dynamic range was per-
formed from 0.0125 to 0.15 ng/μL. Slope, intercept,
and correlation coefficient (r) were determined by
using the least-squares regression analysis form the
calibration curve constructed based on relative peak
area ratios to IS. LOD was evaluated by injection
following dilution with standard solution at the
Fig. 1 Chemical structures of SNAP-8 and IS
Table 1 Comparison of ionic strength between TFA and FA as
additives in the mobile phase
Name Area
0.125 ng/μL(n= 5) 0.5 ng/μL(n=5)
0.1% TFA
SNAP-8 11,916 ± 1171 44,438 ± 1672
IS 30,991 ± 2577 27,287 ± 1558
0.1% FA
SNAP-8 75,020 ± 1636 305,665 ± 3459
IS 242,824 ± 9473 251,054 ± 2408
Ji et al. Journal of Analytical Science and Technology (2020) 11:34 Page 2 of 7

Fig. 2 Optimization of DL temperature in the range 200 to 275 °C
Fig. 3 Mass spectral patterns of SNAP-8 (a) and the IS (b) by LC-MS/MS with ESI mode
Ji et al. Journal of Analytical Science and Technology (2020) 11:34 Page 3 of 7

minimum concentration of calibration range. LOQ of
SNAP-8 was defined as minimum concentration in
the calibration range. The repeatability as relative
standard deviation (%RSD) and the accuracy as rela-
tive error (%RE) were obtained from two different
concentrations from triplicate experiments under the
optimal conditions. The recovery was evaluated by
comparing the percentages of peak area ratio of fil-
tered to that of non-filtered sample (recovery of
100%) including same concentration of SNAP-8.
Liquid chromatography-tandem mass spectrometry
LC-MS/MS analysis was performed in multiple reac-
tion monitoring (MRM) mode using a Shimadzu
Nexera UPLC system (Shimadzu Corp., Kyoto, Japan)
coupled with a LCMS-8050 triple quadrupole mass
spectrometer (Shimadzu Corp., Kyoto, Japan)
equipped with an electrospray ionization (ESI) inter-
face in positive ionization mode. Chromatographic
separation was performed using a betabasic-18 col-
umn (30 mm × 2.1 mm, 5 μm, 150 Å, Thermo Elec-
tron, Waltham, MA) equipped with a betabasic-18
guard column (10 mm × 2.1 mm, 5 μm, 150 Å,
Thermo Electron, Waltham, MA), which separates
polar peptides like SNAP-8 in reverse phase mode.
The column oven was maintained at 40 °C and the
autosampler temperature at 4 °C. Gradient elution was
performed using solvent A (HPLC grade water
Table 2 Ion transitions and collision energy values of SNAP-8 and IS
Analyte Precursor
ion [M +
2H]
2+
(m/
z)
Product ions (m/z)
Transition 1
a
(m/z) CE1
c
(V) Transition 2
b
(m/z) CE2
c
(V) Transition 3
b
(m/z) CE3
c
(V)
Acetyloctapeptide-3 (SNAP-8) 538 102 −30 84 −48 144 −25
Acetylhexapeptide-3 (IS) 445 102 −39 84 −55 144 −30
a
Quantification ion
b
Qualitative ions
c
Collision energy
Fig. 4 MRM chromatograms of the blank spiked with IS (a) and SNAP-8 and IS (b) by LC-MS/MS
Ji et al. Journal of Analytical Science and Technology (2020) 11:34 Page 4 of 7

containing 0.1% FA) and solvent B (ACN containing
0.1% FA) in total flow rate of 0.3 mL/min. Gradient
condition of solvent B was initiated from 0% (0.5 min)
and was increased up to 40% (0.5 to 5 min), and then
held for 1 min (5 to 6 min), and then return to the
initial condition for the next run and hold for 2 min
(6 to 8 min). An aliquot 10 μL of each sample was
injected into the LC-MS/MS system. The mass spec-
trometer interface temperature was maintained at
200 °C, the nebulizing gas flow was at 3.0 L/min, des-
olvation line (DL) temperature at 200 °C, and heat
block temperature at 400 °C. The pressure of
collision-induced dissociation (CID) gas was at 270
kPa. SNAP-8 and IS were successfully detected in the
product-ion scan mode. Quantitative analyses were
performed in the MRM transition mode with quadru-
pole 1 (Q1) and quadrupole 3 (Q3) operated at unit
resolution. A dwell time and pause time were set at
22.0 ms for selected ions.
Result and discussion
Optimal conditions for SNAP-8 analysis by LC-MRM-MS/MS
The effects of FA and TEA as additives in the mobile
phase were investigated. In this result, FA was used due to
higher intensity (about six times) than those of TEA
(Table 1). A C
18
-column (5 μm, 150 Å) with equipped a
guard column was selected for peptide analysis. Analysis
of SNAP-8 was performed using gradient elution within 2
min. The precursor ions of SNAP-8 and IS were selected
with each multiply charged ion in ESI mode. The [M +
2H]
2+
ions of SNAP-8 (m/z 538) and IS (m/z 445) were
selected as precursor multiple ions in positive mode. The
[M + H]
+
ions of SNAP-8 (m/z 1076) and IS (m/z 890)
were detected at low intensities and the [M + 3H]
3+
of
SNAP-8 was not detected. The intensity of SNAP-8 was
monitored with the desolvation line (DL) temperature
from a range of 200 to 275 °C. DL temperature was opti-
mized for [M + 2H]
2+
intensity at 200 °C (Fig. 2). Q3
SCAN spectra of SNAP-8 (a) and IS (b) are shown in Fig.
3. Precursor ions of SNAP-8 (m/z 538) and IS (m/z 445)
were fragmented with collision energy, both product ions
of SNAP-8 and IS were optimized to m/z 102, 84, and
144, respectively. Optimal collision energies were moni-
tored for SNAP-8 (m/z 102 (−30 V), m/z 84 (−48 V), and
m/z 144 (−25 V)) and IS (m/z 102 (−39 V), m/z 84 (−55
V), and m/z 144 (−30 V)). Optimized MRM conditions
are summarized in Table 2and MRM chromatograms of
SNAP-8 and IS are shown in Fig. 4.
Method validation for SNAP-8 analysis by LC-MS/MS
SNAP-8 was detected as a single peak at 1.7 min in ex-
tracts of a blank patch spiked with SNAP-8 standard.
Chromatograms of SNAP-8 in extracts of blank patch
spiked with IS (a), and extracts of blank patch spiked
with SNAP-8 and IS (b) are shown in Fig. 4. The good
linearity was obtained with correlation coefficient (r)of
0.9971 in the range from 0.0125 to 0.1250 ng/mg. LOD
and LOQ were evaluated with diluted standard solution
and their levels were 0.0025 and 0.0125 ng/mg, respect-
ively. Repeatability of 0.05 and 0.1 ng/mg showed 0.20
and 0.02 (%RSD), respectively. Accuracy were 1.44 and −
1.68 (%RE) at 0.05 and 0.1 ng/mg, respectively. The re-
covery showed 76.7% at 0.125 ng/mg of microneedle of
patch (Table 3). In the stability test, SNAP-8 was stable
at least 96 h when this was refrigerated at 4 °C (Table 4
and Fig. 5). Therefore, this method was suitable for
quantification and evaluation test of SNAP in patch
products.
Patch matrix effects for SNAP-8 analysis
Two different calibration curves were compared to as-
sess matrix effects for measurements made using blank
Table 3 Validation data set of SNAP-8 by LC-MS/MS
Analyte Calibration
range (ng/
mg of
microneedle
of patch)
Linearity
(r)
LOD
a
LOQ
b
Repeatability
c
(%) Accuracy
d
(%) Recovery
e
(%)
Added (ng/mg of microneedle of patch)
0.05 0.1 0.05 0.1 0.125
Acetyloctapeptide-
3 (SNAP-8)
0.0125–0.125 0.9971 0.0025 0.0125 0.12 0.02 1.44 −1.68 76.7
a
LOD, limit of detection
b
LOQ, limit of quantification
c
Relative standard deviation (% RSD)
d
Relative error (% RE)
e
Recovery (filtered/non-filtered) × 100
Table 4 Stability test of SNAP-8 in DW at 4 °C
Analyte Concentration of SNAP-8 (0.77 ng/μL)
0 h 24 h 48 h 96 h
SNAP-8 0.75 ± 0.04 0.77 ± 0.03 0.78 ± 0.03 0.75 ± 0.02
Ji et al. Journal of Analytical Science and Technology (2020) 11:34 Page 5 of 7

patches or HPLC grade water, and SNAP-8 in micronee-
dle patches were quantified. Based on the results of four
patches, the calculated SNAP-8 level using HPLC grade
water calibration curve was 89.5 ± 10.5 ng, while it was
76.9 ± 8.6 ng by using matrix calibration curve including
blank patches. In this result, the calculated SNAP-8 level
using matrix calibration curve was 16.3% smaller than
HPLC grade water-based values. Therefore, the matrix
effects of patches must be considered for quantitative
analysis of SANP-8.
Conclusion
Analytical method of SNAP-8 was developed by LC-MS/
MS in MRM mode. In the optimal condition, linearity (r
≥0.9971), repeatability (%RSD = 0.02 to 0.12), accuracy
(%RE = −1.68 to 1.44), and recovery (76.7%) showed
with LOD of 0.0025 ng/mg and LOQ of 0.0125 ng/mg.
When applied to patch product, SNAP-8 was identified
and determined as 76.9 ± 8.6 ng. Therefore, this method
was suitable for determination of SNAP-8 in patch
products, which will be useful for quality control and
evaluation test of SNAP-8 in the cosmetic fields.
Abbreviations
SNAP-25: Synaptosome-associated protein of 25 kDa; SNAP-8: Acetyl
octapeptide-3; BOTOX®: Botulinum toxin; LC-MS/MS: Liquid chromatography-
tandem mass spectrometry; IS: Internal standard; HPLC: High-performance
liquid chromatography; ACN: Acetonitrile; FA: Formic acid;
TEA: Trimethylamine; LOD: Limit of detection; LOQ: Limit of quantification;
r: Correlation coefficient; %RSD: Repeatability; %RE: Accuracy; MRM: Multiple
reaction monitoring; ESI: Electrospray ionization; DL: Desolvation line;
CID: Collision-induced dissociation; Q1: Quadrupole 1; Q3: Quadrupole 3
Acknowledgements
This study was performed at the College of Pharmacy and Research Institute
of Life and Pharmaceutical Sciences, Sunchon National University. This work
was supported by Raphas Co., Ltd. in 2015.
Authors’contributions
MJ and H-SL performed method development, optimization, and validation,
and were a major contributor in writing the manuscript. SC, SJO, and D-YK
performed pre-analytical experiments and sample preparation. YK, CS, JM,
and H-JP performed optimization of method. JDK, DHJ, and M-JP designed
the experiments and supervised this work. All authors read and approved
the final manuscript.
Funding
Not applicable in this section.
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests.
Author details
1
College of Pharmacy, Sunchon National University, Suncheon 540-950,
Republic of Korea.
2
New Drug Development Center, Daegu-Gyeongbuk
Medical Innovation Foundation, Daegu, Republic of Korea.
3
Redone Tech,
Laboratories of Marine New Drugs, Seoul, Republic of Korea.
4
Raphas, Avison
Biomedical Research Center, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, Republic
of Korea.
Received: 20 April 2020 Accepted: 29 July 2020
References
Adler M, Sheridan RE, Deshpande SS, Oyler GA. Neuromuscular transmission
and muscle contractility in SNAP-25-deficient coloboma mice.
Neurotoxicology. 2001;22(6):775–86. https://doi.org/10.1016/S0161-
813X(01)00066-3.
Blanes-Mira C, Clemente J, Jodas G, Gil A, Fernández-Ballester G, Ponsati B,
Gutierrez L, Pérez-Payá E, Ferrer-Montiel A. A synthetic hexapeptide
(Argireline) with antiwrinkle activity. Int J Cosmet Sci. 2002;24(5):303–10.
https://doi.org/10.1046/j.1467-2494.2002.00153.x.
Blasi J, Chapman ER, Link E, Binz T, Yamasaki S, De Camilli P, Südhof TC,
Niemann H, Jahn R. Botulinum neurotoxin A selectively cleaves the
synaptic protein SNAP-25. Nature. 1993;365(6442):160. https://doi.org/10.
1038/365160a0.
Gutiérrez LM, Cánaves JM, Ferrer-Montiel AV, Reig JA, Montal M, Viniegra S. A
peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca2+-
dependent exocytosis in chromaffin cells. FEBS Lett. 1995;372(1):39–43.
https://doi.org/10.1016/0014-5793(95)00944-5.
Fig. 5 Stability test of SNAP-8 for 96 h at 4 °C
Ji et al. Journal of Analytical Science and Technology (2020) 11:34 Page 6 of 7

Gutierrez LM, Viniegra S, Rueda J, Ferrer-Montiel AV, Canaves JM, Montal M. A
peptide that mimics the C-terminal sequence of SNAP-25 inhibits secretory
vesicle docking in chromaffin cells. J Biol Chem. 1997;272(5):2634–9. https://
doi.org/10.1074/jbc.272.5.2634.
Lipotec™. (n.d.) Snap-8, An octave-tide to express yourself at ease. Barcelona,
Spain. https://www.cossma.com/fileadmin/all/cossma/Archiv/ProductInfo/
COS1005_14_ProdSnap8.pdf..
Park J-H, Allen MG, Prausnitz MR. Biodegradable polymer microneedles:
fabrication, mechanics and transdermal drug delivery. J Control Release.
2005;104(1):51–66. https://doi.org/10.1016/j.jconrel.2005.02.002.
Yamauchi PS, Lowe NJ. Botulinum toxin types A and B: comparison of efficacy,
duration, and dose-ranging studies for the treatment of facial rhytides and
hyperhidrosis. Clin Dermatol. 2004;22(1):34–9. https://doi.org/10.1016/j.
clindermatol.2003.11.005.
Zhang S, Qiu Y, Gao Y. Enhanced delivery of hydrophilic peptides in vitro by
transdermal microneedle pretreatment. Acta Pharm Sin B. 2014;4(1):100–4.
Zhou W, Wang PG, Krynitsky AJ, Rader JI. Rapid and simultaneous
determination of hexapeptides (Ac-EEMQRR-amide and H2N-EEMQRR-
amide) in anti-wrinkle cosmetics by hydrophilic interaction liquid
chromatography–solid phase extraction preparation and hydrophilic
interaction liquid chromatography with tandem mass spectrometry. J
Chromatogr A. 2011;1218(44):7956–63.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Ji et al. Journal of Analytical Science and Technology (2020) 11:34 Page 7 of 7