Quantitative Analysis of Pyoluteorin in Anti-fungal Fermentation Liquor of Pseudomonas Species by Capillary Zone Electrophoresis with UV-VIS Detector:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" />
Qiu-Ling Wang1) Xue-Hong Zhang2) Liu-Yin Fan1) Wei Zhang1) Yu-Qian Xu2) Hong-Bo Hu2) Cheng-Xi Cao*[1])
1) Laboratory of Analytical Biochemistry & Bioseparation 2)Laboratory of Applied Microbiology School of Life Science & Biotechnology Shanghai Jiao Tong University Shanghai 200240 China
This er investigated potential utility of capillary zone electrophoresis (CZE) for very succinct but robust quantitative analysis of pyoluteorin (Plt) in anti-fungal fermentation liquor of Pseudomonas species. The experimental conditions for the separation and quantification of Plt were optimized at first. The optimized conditions are: 80 mmol/L pH 8.40 Gly-NaOH buffer 51 cm total length (42 cm effective) and 75 mm i.d. capillary 230 nm wavelength 25 kV 13 mbar 10 sec pressure sample injection and 24oC air-cooling. Under the optimized conditions the migration times of Plt and the internal standard phenobarbital are 2.09 and 2.49 minute respectively the linear response of Plt concentration ranges from 5.0 mg/mL to 1000 μg/mL with high correlation coefficient (r = 0.99977 n = 9) the limits of detection (LOD) and quantification (LOQ) for Plt are 0.66 μg/mL and 2.2 mg/mL the precision values (expressed as RSD) of intra-day and inter-day are 1.19-1.94% and 1.55-6.21% respectively the recoveries of Plt at three concentration levels of 750 250 and 50 mg/ml range from 90.31% to 97.85% and to 98.96% respectively. The developed method can be well used for the quantification of Plt in the fermentation liquor.
Keywords: capillary zone electrophoresis; fermentation; Pseudomonas species; pyoluteorin
Pyoluteorin (Plt) is an antibiotic substance produced by certain strains of Pseudomonas species such as Pseudomonas fluorescens strain Pf-5 CHA0 and fluorescent Pseudomonas sp. M18 [12]. It is composed of a bichlorinated pyrrole
The quantification of Plt in fermentation liquor of Pseudomonas strain CHA0 Pf-5 and M18 could be performed with some methods. Thin
HPCE is one of the most powerful analytical and separative techniques [10-12]. With simple online sample stacking [13-15] its determination sensitivity can reach at the degree of ng/ml even at the level of pg/ml [16] with a normal UV-VIS detector. However no electrophoretic method has been reported for the determination of Plt so far.
Therefore in this work we developed CE method for the quantification of Plt in fermentation liquor of Pseudomonas sp. M18 optimized the parameters affecting the separation and sensitivity of Plt and conducted the investigations on reliability including specificity selectivity linearity LOD LOQ accuracy and precision etc. Herein we reported the very succinct method and results.
Experiments were carried out with an ACS 2000 HPCE apparatus (Beijing Cailu Scientific Inc. Beijing China). The apparatus were comprised of a digital electric power supply (up to voltage 30 kV) a HW-2000 chromatography workstation and an UV-VIS detector that could perform wavelength scanning from 190 to 740 nm. A fused-silica capillary was used (Factory of Yongnian Optical Fiber Hebei China). The capillary was 75 mm I.D.´51 cm (42 cm effective length). I.D. 50 mm capillary was not chosen. Since in the investigation of Plt determination the separation of Plt from others such as MeOH and internal standard could be well performed as shown in Fig. 6. Thus we chose I.D. 75 mm capillary with better sensitivity but not I.D. 50 mm one with poorer sensitivity. An Ultra-pure Water System (SG Ultra Clear system Wasseraufbereitung und Regenerierstation Gmbh Germany) was used to produce ultra pure water with specific conductivity down to 0.055 μS/cm for the analysis of HPCE.
Glycine (biological grade reagent) was purchased from the Shanghai Shisheng Cell and Bio- technology Company (Shanghai China). Sodium hydroxide Hydrochloric acid and methanol (guarantee reagent grade GR) were purchased from the Shanghai Chemistry Reagent Company (Shanghai China). Phenobarbital was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing China). Plt used as standard reagent was purified from fermentation liquor of Pseudomonas species in-house. The purified Plt was analyzed and its purity reaches the chroma pure grade.
The strains studied in this work was sp. M18 which was pre-cultured in a 150 mL conical flask containing 15 mL aliquot of King’s B liquid broth at 28ºC for 12 hours. A portion (8 mL) of this culture was used to inoculate 150 mLl aliquots King’s B medium in 500 mL conical flasks at 28ºC for 60 hours for production of Plt.
Plt as well as phenobarbital can be well purified from fermentation liquor of the strain sp. M18 suspension with ethyl acetate[17]. 600 mL ethyl acetate and 50 mL internal standard (phenobarbital 500 mg/mL) was added into 600 mL culture in one Eppendorf tube then the tube was strongly vibrated for two minutes. After that the mixture in the tube was centrifuged at 5590´g for 5 minutes and Plt was extracted from the culture to the organic phase. The organic phase was transferred to another Eppendorf tube. Then the 600 mL culture was extracted by 300 mL ethyl acetate again. The two organic phases were combined together and dried by vacuum dryness. The residue after dryness in the tube was dissolved with MeOH and diluted to 500 mL with buffer for CE analysis. All solutions were stored at 4oC in a refrigerator until use.
A series of Gly-NaOH buffers with different pH values and concentrations were prepared. The pH values of the buffers ranged from pH 8.0 to pH 11.5 and the concentrations changed from 20 to 200 mmol/L. The stock solution of internal standard was prepared by dissolving 1.0 mg phenobarbital in 2.0 mL MeOH and the concentration was 500 mg/mL.
The sample standard solutions were prepared in accordance with the following procedure. A series of stock solutions with different concentrations of Plt (0.05 0.1 0.2 0.5 1.0 2.5 5 7.5 and 10 mg/mL) were made ahead of schedule adding different concentration solutions of Plt 50 mL and phenobarbital 50 mL into blank fermentation liquor then conducted in accordance with the procedure in Section 2.4. The final concentrations of dilution were 5 10 20 50100 250 500 750 and 1000 mg/mL.
HPCE was conducted as these described below. Before use the new capillary was conditioned by rinsing with 1 mol/L NaOH for 20 min ultra-pure water for 20 min 1 mol/L HCl for 20 min ultra-pure water for 20 min and running buffer for 30 min in order. Between injections the capillary was rinsed with the running buffer for 3.0 min. Detection wavelength was set at the maximum absorbance wavelength of 230 nm detected by an automatic wavelength scanning of Plt in the UV-VIS detector in the CE apparatus. 80 mmol/L pH 8.40 Gly-NaOH buffer was used as background electrolyte. Pressure injection of sample was chosen due to bias of electrokinetic sample injection to Plt as well as phenobarbital. The pressure was set at 13 mbar and the injection time was controlled at 10 sec. The ultimate work voltage was 25 kV. Temperature control of capillary was carried out with 24°C air-cooling. Detection data were collected and processed with the HW-2000 Chromatography Workstation Software.
We firstly optimized the experimental conditions. After than we investigated the validity of developed method. The followings are the results of optimization and investigations.
Applied voltage has very important influence on migration time current strength and resolution (see Fig. S1). The experiments show that the migration time of Plt decreased with the increase of the used voltage. Lower voltage produced longer migration time and poorer peak shape. At 30 kV the migration time decrease greatly but the resolution of peaks reduced possibly due to much high Joule’s heating within the capillary and fast electromigration of analytes. Considering fast determination of Plt and maintain of good resolution 25 kV was chosen as the work voltage as used in the following experiments.
pH value of Gly-NaOH buffer has key importance on migration time and separation of Plt and internal standard As evidently shown in Fig.2 the increase of pH value from pH 8.10 to pH 9.00 led to the decrease of resolution between Plt and phenobarbital but the increase of resolution between MeOH and analyte over pH 8.80 the peak of Plt and internal standard can’t be separated on
Buffer concentration has evident influence on resolution peak shape sensitivity and detection noise especially electric current (see Fig. S3). The results indicate that the resolution among MeOH Plt and internal standard were increased with the increasing of buffer concentration at the same time high concentration of buffer led to much higher current. With the 80 mmol/L buffer the resolution of Plt and phenobarbital were acceptable the current was at a lower level and the peak width of Plt was narrow. Finally 80 mmol/L was chose as the optimized concentration. Our comparative studies showed that 80 mmol/L buffer could supply a quite stable background buffer for the determination of Plt for at least one month.
Finally we achieved the following optimized conditions: 80 mmol/L pH 8.40 Gly-NaOH buffer 51 cm total length (42 cm effective) and 75 mm i.d. capillary 230 nm wavelength 25 kV 13 mbar 10 sec pressure sample injection and 24oC air-cooling.
Validity of a newly developed analytical technique needs to be strictly demonstrated before its application to actual determination of antibiotics. In this work various validation criteria of the developed method of CZE such as specificity precision linearity of response accuracy detection limit (LOD) and quantification limit (LOQ) etc were assessed as described below.
Specificity of a newly developed analytic method should be firstly demonstrated due to its key importance and priority. In the demonstration of this er numerous samples of Plt extracted from the fermentation liquor with ethyl acetate were analyzed repeatedly under different conditions. The purpose of the analyses was to make sure whether or not the peak of target analyte in the electrophograms was exclusive under the given experimental conditions optimized above. We found no interferential peaks appeared in fermentation in different buffer pH value concentration and longer separate time.
Fig. 3 shows the demonstration of exclusive peak of target analyte. Panel A is the electrophoregram of a blank sample of fermentation only MeOH peak can be seen. Panel B unveils the Plt extracted from fermentation liquor without internal standard in buffer. Panel C proves the symmetrical peak of internal standard phenobarbital extracted from fermentation liquor in buffer. Panel D displays the peaks of MeOH Plt and internal standard extracted from the fermentation liquor. All of the experiments discussed above evidently indicate the specificity of the developed CZE method for the determination of targeted analyte.
Linearity of detector response versus concentration of analyte was determined by constructing a calibration curve from a set of standard solutions of Plt extracted from blank fermentation liquor with 9 different concentrations (see Fig. S4). The linear ranged from 5 mg/mL to 1000 μg/mL the correlation coefficient for Plt standards was 0.99977 (n = 9).
Precision of a new method was evaluated by measuring intra-day and inter-day relative standard deviations (RSD) of peak areas ratio between analyte and internal standard extracted from the fermentation liquor. The intra-day values of RSD were calculated
The accuracy was evaluated from a set of recovery experiments performed on samples with three different concentrations. A blank fermentation liquor of Pseudomonas species without Plt was spiked with standard solution of Plt and internal standard of Phenobarbital. The concentrations of Plt in the spiked fermentation liquor were controlled at 50 250 750 μg/mL and the concentration of phenobarbital was 50 μg/mL. The samples used for the recovery runs were prepared as described above (see Section 2.4). The recovery was calculated by the ratio value of Plt and phenobarbital which extracted from the fermentation liquor to the ratio value of them resolving in the run buffer directly. The recovery values of Plt in the spiked samples of fermentation liquor were 98.96 ±2.81% at 50 μg/mL 97.85 ± 6.70% at 250 μg/mL and 90.31 ± 1.00% at 750 μg/mL these values were all within the acceptable range.
It was calculated by setting the signal-to-noise ratio of 3:1 that the limit of detection (LOD) of the developed method was 0.66 μg/mL under 10 sec 13 mbar pressure sample injection. The limit of quantification (LOQ) was computed by use of signal-to-noise ratios of 10:1 that of the developed method was 2.20 μg/mL. A further reduction of LOD and LOQ could be achieved by using long sample injection time but the peaks of Plt and Phenobarbital became wide.
Plt is a new anti-fungal
Acknowledgment: The authors are grateful for the funding provided by the NSFC (No. 20245004 20475036 and 30370041) and the Committee of Science and Technology of Anhui Province (No. 01043905).
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Table 1. The intra-day variations of peak area ratios between Plt and phenobarbital (r = 3 n = 6).
Sample Conc. (μg/mL) | Peak area ratio | SD | RSD (%) |
50 | 0.62 | 0.01 | 1.94 |
250 | 3.19 | 0.04 | 1.19 |
750 | 7.58 | 0.12 | 1.64 |
Table 2. The inter-day variations of peak area ratios between Plt and phenobarbital (r = 3 n = 5).
Sample Conc. (μg/mL) | Peak area ratio | SD | RSD (%) |
50 | 0.58 | 0.04 | 6.88 |
250 | 3.12 | 0.05 | 1.44 |
750 | 7.38 | 0.14 | 1.84 |
Table 3. The recovery of Plt under different concentrations (r = 3 n = 3).
Added Plt (μg/mL) | Peak Areas Ratio of Plt and Phenobarbital | Recovery | |
Extraction from fermentation liquor | Resolving in buffer directly | （％） | |
50 | 0.59 | 0.60 | 98.96±2.81 |
250 | 3.17 | 3.21 | 97.85±6.70 |
750 | 7.39 | 8.18 | 90.31±1.00 |
Fig. 1. Chemical Structure of Pyoluteorin.
Fig. 2. Resolution between MeOH Plt and Phenobarbital as a function of pH value. Conditions: pH 8.10-9.00 Gly-NaOH 25 kV. Other conditions are the same as those in Fig. 2.
Fig. 3. Electrophoregrams of CZE A) Blank sample of fermentation B) Plt extracted from fermentation liquor without internal standard in buffer C) Internal standard Phenobarbital extracted from fermentation liquor in buffer and D) Plt and internal standard extracted from the fermentation liquor in buffer. Conditions: 25 kV 80 mmol/l pH 8.40 Gly-NaOH buffer. Other conditions are the same as those in Fig. 2.
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Online Supporting Materials
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Fig. S1. Influence of applied voltage on migration times of Plt internal standard and MeOH. Conditions: 80 mmol/L pH 8.40 Gly-NaOH buffer 51 cm total length (42 cm effective length) and 75 mm i.d. capillary 230 nm wavelength 13 mbar 10 sec pressure sample injection 24oC air-cooling.
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Fig. S2. Effect of pH value on the migration times of MeOH Plt and phenobarbital. Conditions: pH 8.10~9.00 Gly-NaOH 25 kV other conditions are the same as those in Fig. 2.
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Fig. S3. Effect of buffer concentration on resolution between MeOH plt and internal standard. Conditions: 20~200 mol/l pH 8.40 Gly-NaOH buffer 25 kV. Other conditions are the same as those in Fig. 2.
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Fig. S4. Linearity of peak area ratio of Plt and phenobarbital versus the concentration of Plt.
