ANALYTICAL SCIENCES MAY 2015, VOL.

31 377
2015 © The Japan Society for Analytical Chemistry

Quantification of Lipopeptides Using High-performance Liquid

Chromatography with Fluorescence Detection after Derivatization
Yong MENG, Jin-Feng LIU, Shi-Zhong YANG, Ru-Qiang YE, and Bo-Zhong MU†

State Key Laboratory of Bioreactor Engineering and Institute of Applied Chemistry, East China University of
Science and Technology, Shanghai 200237, P. R. China

A highly sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the
determination of microbial lipopeptides of fluorescent derivatization with 1-bromoacetylpyrene to overcome the limitations
of trace detection of lipopeptides in aqueous solutions. The derivatization of lipopeptides with 1-bromoacetylpyrene was
conducted at 60° C for 20 min under catalysis of triethylamine. The resulting derivative products were separated by HPLC
and determined by a fluorescence detector. Each homolog of lipopeptides in samples was identified by HPLC-MS and
the detection limit after derivatization in an aqueous solution was 2.5 μg/mL (S/N = 3). The calibration curve for
lipopeptides was linear in the concentration range of 0.250 – 4.00 mg/mL. This method has adequate sensitivity and
selectivity for microdetection of lipopeptides in aqueous solutions in mild reaction conditions, which allows this method
to be used in the determination of trace lipopeptides in environmental samples and complex samples.

Keywords Lipopeptide, surfactin, 1-bromoacetylpyrene, derivatization, fluorescence detection

(Received January 19, 2015; Accepted February 25, 2015; Published May 10, 2015)

chromatoplate after hydrolysis and coloration.27,28

Introduction
Lipopeptides are a kind of biosurfactants produced by
microorganisms such as genus Bacillus1,2 and Pseudomonas.3,4
Lipopeptide biosurfactants have received much attention from
both scientific and industrial communities due to their powerful
interfacial and biological activities,5–7 and their great potential
for applications in industrial fields, such as agriculture,8,9
medicine,10–12 cosmetics,13,14 environmental protection15–17 and
the petroleum industry to enhance oil recovery.18–20 Lipopeptides
are a series of structural analogues of different families, and
among them, 23 families covering about 90 lipopeptide
compounds have been reported in the last two decades.21,22 As a
representative member of lipopeptides, surfactin composed of a
peptide chain formed by seven amino acids (L-Glu1–L-Leu2–
D-Leu3–L-Val4–L-Asp5–D-Leu6–L-Leu7) bonded to a hydroxyl fatty
acid chain by lactone bond to form a cyclic lipopeptide.23,24
A number of qualitative methods for the analysis of
lipopeptides in aqueous solutions have been proposed for
screening and separating producing strains.25 The drop-
collapsing test was reported as a rapid method to screen
microorganisms that produce biosurfactants, since drops of cell
suspensions of surfactant-producing microorganisms would
collapse on an oil-coated surface.26 The oil spreading technique
is another rapid detection method to determine the fermentation
broth of microorganisms containing biosurfactants, since the
bacterial colonies would generate clear zones in oil plates.
Thin-layer chromatography (TLC) is a traditional qualitative
method to detect lipopeptides identified by a distinct spot on the
†
To whom correspondence should be addressed.
E-mail: bzmu@ecust.edu.cn
For quantitative analysis of lipopeptides in solutions, the
gravimetric method is a traditional way to determine
lipopeptides,29–31 but it suffers from an inaccuracy in detection of
lipopeptides due to the fact of impurities and loss during the
extraction process, particularly, with low concentrations of
target lipopeptides in solutions. The surface or interfacial
tension measurement method and turbidimetry were also
adopted for quantitative analysis of lipopeptides,32 but were
appropriate only for high concentrations of target lipopeptides
in solutions. Determining the content of dissociative glutamic
in the hydrolyzed samples was another method for measuring
the concentration of lipopeptides,28 but a hydrolytic process was
needed in this method and the veracity of the determination
would be disturbed by dissociative amino acids. High-
performance liquid chromatography (HPLC) has been applied
for qualitative and quantitative analysis of lipopeptides.33,34
However, the reported HPLC method used an ultraviolet detector
(UV-detector) to obtain the ultraviolet absorption signal; the
response of the signal was weak because the ultraviolet
absorption of lipopeptides was caused by the n–π transition of
carbonyls in lipopeptide molecules. The trimethylsilylation gas
chromatography–mass spectrometer (GC-MS) method was a
new way to determine the content of surfactin analogues,22 but
the extraction of the samples and the process of hydrolysis were
complex.
Meanwhile, 1-bromoacetylpyrene (BAP) is a kind of carboxyl-
targeting fluorescence derivatization reagent and it can be
combined with the carboxyls specifically. BAP has been used
for the derivatization of several kinds of carboxylic compounds,
yet it has not been used to determine lipopeptides in aqueous
solutions.33,35,36
This paper presents a quantitative method for the analysis and
378 ANALYTICAL SCIENCES MAY 2015, VOL. 31

determination of surfactins in aqueous solutions. BAP was used

for the derivatization of surfactins to overcome the limitations of
traditional methods and the derivative products were separated
by HPLC and determined by fluorescence detector.

Experimental
Chemicals
A supply of 1-bromoacetylpyrene (BAP, 97%) was purchased
from Sigma-Aldrich (USA) and was used for the derivatization
of surfactins. Acetonitrile (>99.9%) and methanol (>99.9%)
were purchased from J&K (China). Triethylamine (>99.0%),
pyridine (>99.0%), NaCO3 (>99.8%), dodecanoic acid (>98.0%),
myristic acid (>98.0%) and palmitic acid (>98.0%) were
purchased from LingFeng Chemical Reagent Co. (China).
The BAP solution was prepared by dissolving 50.00 mg BAP
in 10 mL acetonitrile and the final concentration of BAP
solution was 5.00 g/L.

Preparation of the standard solutions and samples

The standard surfactin solution was prepared by dissolving the
40.00 mg pure C14-surfactin in 10 mL acetonitrile, and further
dilution was performed by mixing 5.00 mL of the aforementioned
solution in 5.00 mL acetonitrile. The final concentrations of the
standard surfactin solutions were 0.250, 0.500, 1.00, 2.00 and
4.00 g/L. The standard surfactin samples were used to make the
calibration curves.
The pure surfactin samples including C13-surfactin, C14-
surfactin and C15-surfacin were purificated by preparative
HPLC,37 where C13-, C14- or C15-refers to the number of the
carbon atom of β-hydroxyl fatty acid of surfactin molecule.
The crude samples of surfactins were isolated from certain
volumes of a cell-free broth of Bacillus subtilis HSO 121 by
acid precipitation and organic extraction.37
Derivatization method
The surfactin samples extracted from 10.0 mL cell-free broth,
2.00 mg BAP and 0.100 mL triethylamine were mixed and
dissolved in acetonitrile, and the final volume of the reaction
mixture was 1.00 mL. The solution was heated for a given time.
Then, 1.00 mg pure C14-surfactin, 1.00 mg BAP and 0.100 mL
triethylamine were mixed and dissolved in acetonitrile, and the
final volume of the reaction mixture was 1.00 mL. The solution
was heated at 60° C for 20 min. Pure C13-surfactin sample and
pure C15-surfactin sample were reacted via the same method.
Next, 10.0 mg dodecanoic acid, 20.0 mg BAP and 1.00 mL
triethylamine were mixed and dissolved in acetonitrile and the
final volume of the reaction mixture was 15.0 mL. The solution
was heated at 60° C for 20 min. Myristic acid and palmitic acid
were reacted via the same method.
For the samples for the contrasting test, the surfactin sample
extracted from 10 mL cell-free broth, 2.00 mg BAP and
0.100 mL triethylamine were mixed and dissolved in acetonitrile
and the final volume of the reaction mixture was made up to
1.00 mL. The solution was heated at 60° C for 20 min with air
pumped in continuously by an air pump.
Finally, 100 μL standard C14-surfactin sample of different
concentration was mixed with 100 μL triethylamine and 200 μL
BAP acetonitrile solution, and the solution was heated at 60° C
for 20 min.
HPLC/HPLC-MS analyses
The HPLC system (Shimadzu, Japan) consisted of two pumps,
a column oven, a sample injector, a UV detector, and an RF-
Fig. 1 a. Preparative RP-HPLC of surfactin mixtures (chromato-
graphic peaks: 1, C13-surfactin; 2, iso C14-surfactin; 3, n C14-surfactin;
4, C15-surfactin). b. Chromatogram of derivative surfactin sample
(chromatographic peaks: 1, D-C13; 2, D-C14; 3, D-C15).
20A prominence fluorescence detector (Shimadzu, Japan). The
analytical HPLC column was a C18 reversed phase column,
5 μm, 250 × 4.6 mm (Elite, China). The HPLC-MS system
(Agilent 1100 Liquid Chromatography-LCQ Deca XP Ion Trap
Mass) was used to analyze the products after derivatization.
Methanol (A) and redistilled water (B) were mixed as the
mobile phase. The gradient (A/B) was maintained at 80% in the
first 2 min, then from 80 to 100% for the next 18 min, and
maintained at 100% after 20 min. HPLC analysis of the BAP
derivatives of surfactin was carried out with a flow-rate of
1.0 mL/min at 30° C. Then, 10.0 μL derivative sample was
injected into the HPLC equipment. The excitation wavelength
(λex) set in the fluorescence detector was 366 nm and the
emission wavelength (λem) was 420 nm.35
The MS instrument connected with the HPLC system was
operated in the positive mode and its capillary voltage, sample
cone voltage and extraction cone voltages were 3 kV, 100 and
6 V, respectively. The scanned area was 200 – 2000 m/z.
Results and Discussion
Derivatization of surfactins with 1-bromoacetylpyrene
Surfactin samples were determined after derivatization with
BAP and each homolog of surfactins was identified. The major
components of crude surfactin samples after derivatization are
shown in Fig. 1b and the mass spectrometry information of each
derivative surfactin (D-surfactin) homolog is shown in Fig. 2.
The surfactins produced by Bacillus subtilis HSO 121 were
mainly constituted from C12-surfactin to C17-surfactin. However,
C13-surfactin, C14-surfactin and C15-surfactin were the principal
components.22,37 After derivatization with BAP, surfactins were
reacted with a fluorophore by nucleophilic substitution
mechanism, and it greatly improved the sensitivity of analysis
ANALYTICAL SCIENCES MAY 2015, VOL. 31 379

Fig. 2 MS spectrum of D-surfactin. a, b, c are MS of peaks 1, 2, 3 in Fig. 1b.

by HPLC. The chromatogram of derivative products is shown

in Fig. 1b. Compared with the chromatogram of major
components in surfactin samples by UV-detector (Fig. 1a37), it
could be confirmed preliminarily that the peaks of components
ranging from 15.0 to 20.0 min were D-surfactins by comparing
the shape of the chromatographic peaks between Figs. 1a and
1b. The retention times of each surfactin homolog were
confirmed via the detection of pure surfactin samples with the
same method. By comparing the chromatograms of crude
surfactin samples and pure surfactin samples, the retention times
of derivative C13-surfactin (D-C13), derivative C14-surfactin
(D-C14) and derivative C15-surfactin (D-C15) were confirmed to
be 15.8, 17.3 and 18.8 min, respectively. It was further
concluded that D-surfactins were identified in Fig. 1b. As
C14-surfactin contained iso and n types of surfactin isoforms,22,38
the two peaks labeled by 2 in Fig. 1b are iso and n types of
D-C14 isoforms. It was known that the molecular mass of these
three surfactin homologs were m/z 1007.68, 1021.68 and
Fig. 3 Stability of derivative products.
1035.68.38 The relative molecular mass of BAP was m/z 323.18.
HPLC-MS was utilized to verify whether the three components
were the D-surfactins. The structure of a surfactin molecule has
two carboxyls; the mass spectrometry information (Fig. 2)
revealed only one carboxyl was reacted with BAP. The contents of D-surfactins over time are shown in Fig. 3, which
molecular ion peaks of m/z 1250.4, 1264.5 and 1278.6 showed implied the derivative products were stable in 12 h after
the relative molecular mass of three D-surfactin homologs. derivatization.
D-surfactin samples were placed at 25° C hermetically and
Stability of derivative products detected by HPLC after 12, 24 and 48 h. The derivative products
The stability of derivative products was investigated and the may decompose after derivatization but the reduction of
380
Fig. 4 Optimization of derivatization reaction time and temperature.
derivative products was not apparent in 12 h. Of the total of
derivative products, about half was left after 24 h and one third
after 48 h.
Interference factors of derivatization
There were not many impurities in surfactin samples that
would interfere with the derivatization after acid precipitation
and organic reagent extraction.22,28 The fatty acids, which have
a similar length of the carbon chain as surfactins, were the only
impurities in the reaction mixture. Dodecanoic acid, myristic
acid and palmitic acid were selected as reactants in the
derivatization with BAP in order to optimize reaction conditions.
By comparing the retention time of derivative dodecanoic acid,
myristic acid and palmitic acid with D-surfactin, it was found
that the fatty acids with similar length of the carbon chains as
surfactins have no effect on the determination of surfactin. The
retention times of derivative dodecanoic acid, myristic acid and
palmitic acid were 25.5, 30.9 and 35.9 min, respectively, which
were considerable longer than the retention times of D-surfactins.
There are a few interfering factors that could lead to the
quenching of fluorescent reagents. The role of oxygen was
investigated to confirm whether it would act as a quencher of
BAP to obstruct the determination of surfactins. Peak areas of
D-C14 were compared between normal reactions of crude
surfactin samples with reaction in air injection conditions. The
results showed that the fluorescence intensity of D-surfactin
showed no differences between the two reaction conditions. As
such, there was no need to remove oxygen in the reaction system
or to ensure special protection.
Optimum conditions of derivatization
Several reaction factors were examined during the optimization
studies of the derivatization, including reaction time, temperature
and catalyzer. The extent of derivatization over time and
temperature are shown in Fig. 4. The optimum reaction time
was confirmed to be 20 min and the optimum reaction
temperature was confirmed to be 60° C. The most suitable
catalyzer for the derivatization was triethylamine.
The peak areas of D-C14 were used to assess the extent of
reaction. Initially, 60°C was selected as the reaction temperature
and 10 min, 20 min, 30 min, 1 h and 3 h were selected as the
reaction times. The relation of D-C14 peak areas with reaction
time is shown in Fig. 4a. The peak areas increased from 0 to
20 min, but the peak areas declined with the extension of
reaction time. In view of the derivatization, this method was a
ANALYTICAL SCIENCES MAY 2015, VOL. 31
kind of nucleophilic substitution; the derivatization was a
reversible reaction and the optimum reaction time was confirmed
to be 20 min. Then, 30, 40, 50, 60 and 70° C were selected as
the reaction temperatures. Figure 4b shows that although peak
areas increased with the rise of temperature, it remained about
the same above 60° C. Therefore, the temperature accelerated
the reaction rate, but as the reaction time was confirmed to be
20 min, the derivatization reacted towards completion in 60° C.
Considering the boiling point of acetonitrile is 80° C, high
temperatures would influence the volume of the reaction
mixture. The optimum reaction temperature was confirmed to
be 60° C. The derivatization was a nucleophilic substitution and
a monomolecular HBr liberated from the reaction system. As a
result, an alkaline catalyzer was necessary. The conventional
alkaline catalyzers pyridine and Na2CO3 were investigated in
addition to the original catalyzer triethylamine. Comparing the
D-surfactin peak areas after derivatization catalyzed by pyridine,
Na2CO3 and triethylamine shows the fluorescence intensity of
D-surfactin catalyzed by pyridine was half of that catalysis by
triethylamine, and no derivative products were detected after the
catalysis of Na2CO3. Therefore, triethylamine was selected as
the most suitable catalyzer for the derivatization.
Calibration curves and the detection limit
Calibration curves have been determined according to the
linear relations of surfactin concentration and fluorescence
intensity. The detection limit calculated from the calibration
curves with S/N = 3 was 2.5 μg/mL.
Standard C14-surfactin samples with concentrations of 0.250,
0.500, 1.00, 2.00 and 4.00 g/L were reacted in optimized
conditions. The relative formula of C14-surfactin contents and
peak areas was y = 1.252x – 0.385 (Formula-1), and the relative
formula of C14-surfactin contents and peak heights was y =
0.251x – 0.006 (Formula-2). The mass of different surfactin
homologs in samples were consequently determined according
to the calibration curves.
Blank samples that used isometric acetonitrile to replace
C14-surfactin were determined seven times. Heights of baseline
noise at 15 to 17 min were recorded. With the S/N = 3, three
times the highest height of baseline noise was substituted in
Formula-2. The content of C14-surfactin was determined by
calculation and was regarded as the detection limit. As the
highest height of baseline noise was 0.012 mV, it could be
confirmed that the detection limit of surfactin mass in the
injected sample was 0.025 μg. Thus, the detection limit in the
ANALYTICAL SCIENCES MAY 2015, VOL. 31 381

Fig. 5 Chromatograms of surfactin sample before and after derivatization (a: chromatogram of
surfactin sample detected by UV-detector before derivatization; b: chromatogram of surfactin sample
detected by UV-detector after derivatization; c: chromatogram of surfactin sample detected by
fluorescence-detector before derivatization; d: chromatogram of surfactin sample detected by
fluorescence-detector after derivatization).

derivatization system of this method was 2.5 μg/mL (S/N = 3).

Quantitative determination of surfactin samples
A surfactin sample was derivatized by BAP in 1 mL reaction
system. The chromatograms of surfactin sample before and
after derivatization are shown in Fig. 5. Figures 5a and 5c show
the chromatograms detected by UV detector and fluorescence
detector before derivatization, respectively, which indicate the
surfactin was hardly detected. Figures 5b and 5d show the
chromatograms after derivatization, which indicate that detection
sensitivity drastically improved after derivatization.
Whole peak areas of D-C13, D-C14 and D-C15 were 3.92 ×
107 μV*s. The content of surfactins in injection could be
calculated after substituting the peak area in Formula-1. The
content of surfactins in the injected sample worked out as
31.62 μg and the content of surfactins in the sample worked out
as 3.16 mg.
This method could be used to determine not only surfactins
but also almost all families of lipopeptides. On condition that a
family of lipopeptides whether cyclic lipopeptide (surfactin,
lichenysin, iturin, etc.) or linear lipopeptide (spiroidesin, etc.)
has carboxyls in their molecules,21 BAP or other applicable
fluorescence derivatization reagents could be used for
derivatization and determination could be performed by a
fluorescence detector.
Conclusions
In this work, a highly sensitive and selective high-performance
liquid chromatographic method has been developed for the
determination of biosurfactant surfactins with fluorescent
derivatization using 1-bromoacetylpyrene in aqueous solutions,
and a low detection limit of 2.5 μg/mL (S/N = 3) was
successfully achieved. According to the peak area integral value
in the liquid chromatogram and compared with the calibration
curves, the content of each homolog of surfactins can be
determined. This method could be generalized to determine
almost all families of lipopeptides bearing carboxyl groups in
their molecules and applied to analyze environmental samples
and complex samples.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant No. 21203063) and the 863 Program
(Grant No. 2013AA064403).
382
References
1. J. Vater, B. Kablitz, C. Wilde, P. Franke, N. Mehta, and S.
S. Cameotra, Appl. Environ. Microbiol., 2002, 68, 6210.
2. S. Fahim, K. Dimitrov, F. Gancel, P. Vauchel, P. Jacques,
and I. Nikov, Bioresour. Technol., 2012, 126, 1.
3. J. T. d. Souza, M. d. Boer, P. d. Waard, T. A. v. Beek, and J.
M. Raaijmakers, Appl. Environ. Microbiol., 2003, 69, 7161.
4. H. Rokni-Zadeh, W. Li, E. Yilma, A. Sanchez-Rodriguez,
and R. D. Mot, Environ. Microbiol. Rep., 2013, 5, 160.
5. F. Peypoux, J. M. Bonmatin, and J. Wallach, Appl.
Microbiol. Biotechnol., 1999, 51, 553.
6. I. M. Banat, A. Franzetti, I. Gandolfi, G. Bestetti, M. G.
Martinotti, L. Fracchia, T. J. Smyth, and R. Marchant, Appl.
Microbiol. Biotechnol., 2010, 87, 427.
7. G. Seydlová and J. Svobodová, Cent. Eur. J. Med., 2009, 3,
123.
8. D. P. Sachdev and S. S. Cameotra, Appl. Microbiol.
Biotechnol., 2013, 97, 1005.
9. H. Tran, A. Ficke, T. Asiimwe, M. Höfte, and J. M.
Raaijmakers, New Phytol., 2007, 175, 731.
10. L. BenMohamed, S. Wechsler, and A. Nesburn, Lancet
Infect. Dis., 2002, 2, 425.
11. R. H. Baltz, V. Miaoa, and S. K. Wrigleya, Nat. Prod. Rep.,
2005, 22, 717.
12. J. N. Steenbergen, J. Alder, G. M. Thorne, and F. P. Tally, J.
Antimicrob. Chemother., 2005, 55, 283.
13. C.-L. McGregor, L. Chen, N. C. Pomroy, P. Hwang, S. Go,
A. Chakrabartty, and G. G. Privé, Nat. Biotechnol., 2003,
21, 171.
14. X. Wang, G. Huang, D. Yu, B. Ge, J. Wang, F. Xu, F.
Huang, H. Xu, and J. R. Lu, PLoS One, 2013, doi: 10.1371/
journal.pone.0076256.
15. L.-M. Whang, P.-W. G. Liu, C.-C. Ma, and S.-S. Cheng, J.
Hazard. Mater., 2008, 151, 155.
16. C. Mulligan, R. Yong, and B. Gibbs, J. Soil Contam., 1999,
8, 231.
17. A. K. Singh and S. S. Cameotra, Environ. Sci. Pollut. Res.,
2013, 20, 7367.
18. J. F. B. Pereira, E. J. Gudiña, R. Costa, R. Vitorino, J. A.
Teixeira, J. A. P. Coutinho, and L. R. Rodrigues, Fuel,
ANALYTICAL SCIENCES MAY 2015, VOL. 31
2013, 111, 259.
19. M. Bao, Y. Pi, L. Wang, P. Sun, Y. Li, and L. Cao, Environ.
Sci.: Processes Impacts, 2014, 16, 897.
20. N. Youssef, D. R. Simpson, M. J. McInerney, and K. E.
Duncan, Int. Biodeterior. Biodegrad., 2012, doi: 10.1016/j.
ibiod.2012.05.010.
21. X. Y. Liu, S. Z. Yang, and B. Z. Mu, Biotechnol. Bull.,
2005, 18.
22. Y. Zhao, S. Z. Yang, and B. Z. Mu, Anal. Sci., 2012, 28,
789.
23. S. Z. Yang, D. Z. Wei, and B. Z. Mu, J. Biochem. Biophys.
Methods, 2006, 68, 69.
24. S. Z. Yang, D. Z. Wei, and B. Z. Mu, J. Biochem. Biophys.
Methods, 2007, 70, 519.
25. N. H. Youssef, K. E. Duncan, D. P. Nagle, K. N. Savage, R.
M. Knapp, and M. J. McInerney, J. Microbiol. Methods,
2004, 56, 339.
26. D. K. Jain, D. L. Collins-Thompson, H. Lee, and J. T.
Trevors, J. Microbiol. Methods, 1991, 13, 271.
27. T. Matsuyama, M. Sogawa, and I. Yano, Appl. Environ.
Microbiol., 1987, 53, 1186.
28. T. Chen, S. Z. Yang, and B. Z. Mu, Oilfield Chem., 2004,
21, 385.
29. N. I. A. Haddad, J. Wang, and B. Z. Mu, Protein Pept. Lett.,
2009, 16, 7.
30. M. J. McInerney, M. Javaheri, and D. P. N. Jr, J. Ind.
Microbiol., 1990, 5, 95.
31. H. L. Chen and R. S. Juang, Biochem. Eng. J., 2007, 38, 39.
32. H.-H. Suh, G.-S. Kwon, C.-H. Lee, H.-S. Kim, H.-M. Oh,
and B.-D. Yoon, J. Ferment. Bioeng., 1997, 84, 108.
33. S. C. Lin, Y. C. Chen, and Y.-M. Lin, J. Chromatogr. A,
1998, 825, 149.
34. C. Sivapathasekaran, S. Mukherjee, R. Samanta, and R.
Sen, Anal. Bioanal. Chem., 2009, 395, 845.
35. S. S. Kelly, A. G. Bishop, E. P. Carmody, and K. J. James,
J. Chromatogr. A, 1996, 749, 33.
36. H. Ochiai, N. Uchiyama, K. Imagaki, S. Hata, and T.
Kamei, J. Chromatogr. B, 1997, 694, 211.
37. X. Y. Liu, S. Z. Yang, and B. Z. Mu, J. Pept. Sci., 2008, 14,
864.
38. X. Y. Liu, N. I. A. Haddad, S. Z. Yang, and B. Z. Mu,
Protein Pept. Lett., 2007, 14, 766.