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Development of a Reversed-Phase Liquid Chromatographic Assay for the Quantification of Total Persipeptides in Ferment.... Article in Chromatographia · July 2016 DOI: 10.1007/s10337-016-3140-y
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Chromatographia (2016) 79:1325–1332 DOI 10.1007/s10337-016-3140-y
ORIGINAL
Development of a Reversed‑Phase Liquid Chromatographic Assay for the Quantification of Total Persipeptides in Fermentation Broth Fatemeh Mohammadipanah1,2 · Hamed Kazemi Shariat Panahi1,2 · Fatemeh Imanparast3 · Javad Hamedi1,2
Received: 11 January 2016 / Revised: 2 July 2016 / Accepted: 6 July 2016 / Published online: 28 July 2016 © Springer-Verlag Berlin Heidelberg 2016
Abstract The emergence and prevalence of multi-drugresistant bacterial strains increase the potential for outbreaks of incurable infections. The discovery of novel antibiotics and pharmacological preparations requires the identification of novel bioactive small molecules. A specific, sensitive, and reliable quantification method using high-performance liquid chromatography (HPLC) with UV detection was developed for the determination of total persipeptides (A and B), which are cyclic pentapeptides found in the fermentation broth of Streptomyces zagrosensis UTMC 1154 that exhibit bioactivity against methicillin-resistant Staphylococcus aureus (MRSA). A simple liquid–liquid extraction (LLE) method using butanol was employed to extract persipeptides from the fermentation broth prior to HPLC analysis. The chromatographic separation of persipeptides and the internal standard, virginiamycin, was achieved with a gradient of acetonitrile and water on a C18 reversed-phase analytical column in a 25-min analytical run utilizing a flow rate of 0.8 mL min−1 and detection at 210 nm. The whole assay was validated,
Electronic supplementary material The online version of this article (doi:10.1007/s10337-016-3140-y) contains supplementary material, which is available to authorized users. * Fatemeh Mohammadipanah
[email protected] 1
Department of Microbial Biotechnology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, 14155‑6455 Tehran, Iran
2
Microbial Technology and Products Research Center, University of Tehran, 14155‑6455 Tehran, Iran
3
Department of Medical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
and the method presented a linear response range with a regression coefficient of determination R2 of 0.9996 for the quantification of persipeptides in the concentration range of 3.9–250.0 µg mL−1, as well as extraction recoveries ranging from 54.78 ± 9.83 % to 56.45 ± 16.33 %. The bias and the precision of the proposed method were 2.0 in general
Theoretical plates per column (N)
Efficiency at 10 % was 20,859.68 Efficiency at 50 % was 20,593.37
>2000
Tailing/asymmetry factor
1.013
Capacity factor (k′)
5.35
≤2.0 >2
System Suitability Testing
Table 2 Accuracy and precision of the persipeptides assay developed in the present work
System suitability testing (SST), an integral component of HPLC methods, is performed to verify that the resolution and reproducibility of the chromatographic system are adequate for the intended analysis [23]. This is accomplished before verifying the accuracy and precision of the HPLC data collected. Transferring a gradient method between laboratories, implementing a published gradient method, or even transferring such a method from one instrument to another within the same laboratory can be a challenging process [15] due to differences in dwell volume and gradient distortion [15]. The dwell volume of the Cecil instrument employed in this study was calculated to be 1.13 mL (additional data are available in Online Resource 3 in the ESM). The flow rate was found to be consistently within 1 % of the set point by measuring a mobile phase volume of 10 mL using a stopwatch. Therefore, the instrument was shown to be able to reliably perform the desired gradient during the assay (additional data are available in Online Resource 4 in the ESM). Other significant parameters in the final HPLC method developed to assay for persipeptides were calculated and compared with the US Food and Drug Administration’s (FDA) Center for Drug Evaluation and Research (CDER) guidelines [18] (Table 1).
Nominal concentration (µg mL−1)
Accuracy (%)
RSD (%)
125.0 15.6
100.5 ± 1.4 99.1 ± 6.5
1.3 6.5
3.9
103.1 ± 9.3
9.0
HPLC Method Validation The method based on HPLC coupled with UV detection was finally approved by evaluating the standard calibration curve, ILOQ and MLOQ, accuracy, precision, recovery, and stability of the method and comparing them with FDA guidelines [24] (unless otherwise stated). Standard calibration curves for persipeptides were obtained using seven calibrators over the linear range 3.9–250 μg mL−1 (i.e., 3.9, 7.8, 15.6, 31.2, 62.5, 125, and 250 μg mL−1). Linear least square regression analysis of the persipeptides/virginiamycin peak area ratio (virginiamycin was the internal standard) versus the persipeptides concentration was performed; a regression equation of y = 0.0127x + 0.0221 was obtained with a coefficient
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n = 5 at each concentration level
Table 3 Recovery of persipeptides using the LLE method developed in this work Nominal conc. (µg mL−1)
Standard sample response (peak area)
NSFS response (peak area)
SFS response Recovery (%) (peak area)
7.8
166.7
205.3
296.6
54.78 ± 9.83
15.6
337.9
396
56.45 ± 16.33
n = 3 at each concentration level NSFB nonspiked fermentation sample (n = 4), SFB spiked fermentation sample (n = 4)
of determination (R2) of 0.9996. The LOD and LOQ for persipeptides were calculated as 27 and 83 μg L−1, respectively. Three concentration levels (low: 3.9, intermediate: 15.6, and high: 125 μg mL−1) were employed to determine the accuracy and precision of the proposed assay. Five injections of each persipeptides standard were carried out. The accuracy of the method was 99.1–103.1 % and the precision (RSD) was 1.3–9.0 % (Table 2). Samples taken from the same fermentation broth that were spiked, or not, with the persipeptide standard underwent extraction using the sample preparation method described earlier in this paper, and then HPLC analysis was carried out. The results are summarized in Table 3. Stability The stability of standard persipeptides in fermentation culture medium under various conditions was investigated. Virginiamycin, used as the internal standard in this study, is
Development of a Reversed-Phase Liquid Chromatographic Assay for the Quantification of Total…
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Table 4 Effects of different preservation periods and conditions on persipeptides stability Preservation conditions
Preservation period (h)
Persipeptides/IS Percentage of peak area ratio the persipeptides that were degradeda
Room temperature
0 3 6 9 24 33 48 24 33 48 0 24
0.41 0.41 0.38 0.36 0.32 0.30 0.27 0.33 0.33 0.31 0.33 0.29
4 °C
LLE and preservation in BuOH at 4 °C One freeze–thaw cycle
– 0 7.3 13 21 27 34 19 19 27 – 13
0
0.33
–
24
0.31
6
a
Difference between the signals recorded for the persipeptides peak at the start and at the end of storage under the conditions noted (%)
Table 5 Experimental values of the standardized effect, sum of squares, and % contribution for each of the factors analyzed Factor
Standardized effect
Sum of squares % Contribution
Organic solvent %
−306.10
281,100
26.82
Flow rate Dummy 1 Dummy 2 Column temp. Slope of gradient
−75.93 165.77 351.67 −41.43 −145.90
17,297.61 82,435.76 371,000 5150.16 63,860.43
1.65 7.87 35.40 0.49 6.09
Dummy 3 Dwell volume Detection wavelength
28.23 5.33 −52.57
Time constant
−177.90
Dummy 4
−201.13
2391.36 85.33 8289.76 94,945.23 121,400
0.23 0.008 0.79 9.06 11.58
very stable in the alkaline pH (8.5–9) of the culture medium. Additionally, virginiamycin was previously shown to be highly stable in culture medium incubated at 25–35 °C for 72 h [25]. Therefore, we can assume that the degradation of virginiamycin during preservation at room temperature or lower as well as in a matrix medium with an alkaline pH is negligible. In accordance with this, the UV-detected peak
Fig. 3 Half-normal probability plot – no significance effect of investigated parameters on the persipeptide peak area was observed
areas of virginiamycin in different chromatograms were consistent with each other. The implementation of virginiamycin as a stable IS allowed us to check for any other possible erroneous operations, including inaccuracy following LLE or/ and sample injection. It can be assumed from Table 4 that the concentration of persipeptides remained relatively constant during 3 h of sample storage at room temperature, and only minimal (nonsignificant) degradation (a 7.3 % decrease) of the analyte in question was observed at 6 h. Nine hours of storage at room temperature led to a 13 % loss of persipeptides, whereas 24 hours of storage resulted in a degradation of 21 %. After 33–48 h, a 27–34 % drop in persipeptide content was observed. Interestingly, the degradation of persipeptides was the same regardless of whether the samples underwent extraction immediately after harvesting the fermentation broth following storage at 4 °C for 24 h (13 %) or the storage of whole fermentation broth at room temperature for 9 h (13 %) (Table 5). Preservation of the fermentation broth at 4 °C for 24–33 h resulted in considerable loss of persipeptides: 19 %, which increased to 27 % at 48 h. However, one freeze–thaw cycle during 24 h of preservation did not damage the persipeptides (6 % degradation). In conclusion, short-term storage of either the whole fermentation broth at ambient temperature or the preservation of its extract at 4 °C were found to be acceptable in terms of persipeptides degradation, but maintenance at −20 °C is preferable when it is necessary to store the persipeptides in BuOH for longer periods. Robustness Deliberately swapping the values of each parameter between two investigation levels was shown to have virtually no effect on the resulting chromatogram; there was
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almost no change in response (i.e., peak area; see Fig. 3). This indicates that the proposed HPLC method is robust with respect to the investigated parameters in the range tested (see the section “Robustness Test of the HPLC Method” and Online Resource 5 in the ESM).
Conclusions In this paper, we have described the validation of an HPLC method for quantifying persipeptides. This method can be performed rapidly, requiring as little as 25 min; it is simple to carry out, with no need for an extensive work-up; it is robust with respect to the ranges of many parameters; and it yields reliable and reproducible results. The proposed method can be applied in routine assays to determine the concentration of total persipeptides in fermentation broths. A rapid, reliable, and simple assay of these potential chemotherapeutic agents is a valuable tool given the bioactivity of persipeptides against MRSA. An analytical method is a fundamental requirement for any drug discovery process. It may be applied, for example, in fermentation optimization of S. zagrosensis in an attempt for obtaining large amount of persipeptides which is required during early early stages of drug discovery program. The structures of persipeptides suggest some other potent pharmaceutical activities that can be investigated more accurately and appropriately if persipeptides amount could be determined by any approved analytical method.The developed method may also be modified to include preparative rather than analytical HPLC, thus allowing pure persipeptides to be obtained more rapidly and efficiently than previously reported methods. Compliance with Ethical Standards This article does not contain any studies with human or animal subjects performed by any of the authors. Conflict of interest No potential conflict of interest is reported by the authors.
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