Quantification of Lipopeptides Using High-performance Liquid

ANALYTICAL SCIENCES MAY 2015, VOL. 31

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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)

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 dropcollapsing 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: [email protected]

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chromatoplate after hydrolysis and coloration.27,28 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. Highperformance 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 carboxyltargeting 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

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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, C14surfactin 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 (chromatographic 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


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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 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 molecular ion peaks of m/z 1250.4, 1264.5 and 1278.6 showed the relative molecular mass of three D-surfactin homologs. Stability of derivative products The stability of derivative products was investigated and the

Fig. 3 Stability of derivative products.

contents of D-surfactins over time are shown in Fig. 3, which implied the derivative products were stable in 12 h after derivatization. D-surfactin samples were placed at 25° C hermetically and detected by HPLC after 12, 24 and 48 h. The derivative products may decompose after derivatization but the reduction of

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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 C. The most suitable temperature was confirmed to be 60° 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

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


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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).

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