Understanding HPLC and Its Selection for the Detection and Quantification of Iturin A

High Performance Liquid Chromatography (HPLC) is a technique that separates compounds (analytes) dissolved in a liquid, mobile phase by using their specific interaction with a stationary phase. It operates on the principle of affinity-driven partitioning of compounds between the mobile phase and the stationary phase. The K value or partition coefficient for a particular compound is directly proportional to the affinity of the analyte to the stationary phase. On injection into the column, the compounds with a higher affinity and thus K value for the stationary phase are eluted more slowly than those with less as the interactions between said compound and the stationary phase hinder their movement.

HPLC is advantageous over other chromatographic techniques mainly due to its sensitivity and selectivity. HPLC can detect even trace amounts of the analyte, from nanograms to picograms. HPLC is also beneficial for its versatility as it can separate a variety of products ranging from polar and nonpolar compounds, acidic and basic compounds, and large and small molecules ​[1].

From literature surveys, it was found that Reverse Phase HPLC is the standard method used for the identification, quantification, and purification of Iturin A and its isoforms ​[2][3]. The​ specifics of HPLC for Iturin A have been specified in various papers ​[4]. However, the conditions to obtain a proper chromatogram for your sample through HPLC are extremely system-specific; therefore, the conditions needed for our elution of Iturin A in our HPLC system had to be optimized. This is known as the method file construction and was done via multiple iterations.

Method File Construction

Preparation of Standard Samples

A 1:1 dilution series of pure Iturin A samples was prepared in the range 100µg/ml to 1.5625µg/ml. A dilution range with lower concentration values was preferred for the standard curve as the concentration of Iturin in our samples will be lower. The specifics of the preparation of the dilution series are shown in the table below:

Sample Number Concentration (µg/ml) Total Volume (ml) Iturin Stock Volume (µl) Methanol (µl) Remaining (µl)
200g/ml 800 500
9 100 1 ml 500 µl 500 nil
8 50 1 ml 500 µl 500 nil
7 25 1 ml 500 µl 500 nil
6 12.5 1 ml 500 µl 500 nil
5 6.25 1 ml 500 µl 500 nil
4 3.125 1 ml 500 µl 500 nil
3 1.5625 1 ml 500 µl 500 500 µl
2 Blank Crude methanol 1 ml
1 Blank HPLC grade methanol 1 ml

Preparation of Instrument

A C-18 column with a 5µm pore size was fitted with a guard column at the bottom, and the entire column was placed into the oven of the HPLC machine.

All the mobile phase solvents mentioned in literature—0.1% TFA in water, acetonitrile, and methanol—were prepared or obtained and ultrasonicated before connecting them with the pumps.

The instrument was washed with water and acetonitrile before starting the method file development process.

HPLC Runs to Obtain the Ideal Method File

100µl of each sample from the Iturin dilution series was added into vials and loaded onto the HPLC autosampler. The concentration of 12.5 µg/mL was primarily selected for method development, as it represents a median value that is neither too high nor too low.

Nine HPLC runs were performed using the 12.5µg/ml sample, and three were performed using the 25µg/ml sample. The 25µg/ml was tested for three runs to check if the instrument detects only higher concentrations of Iturin A.

The parameters were set on the computer system connected to the instrument.

Various concentrations of methanol, acetonitrile, and 0.1% TFA in water were tested for the mobile phase.

Note: The column is purged if the mobile phase is changed between each run. Before purging, the solvent is put through an ultrasonicator to remove any air bubbles that may cause blockage in the column if not addressed.

Two flow rates were tested: 0.8 ml/min and 1 ml/min.

Various iterations of parameters were run to obtain the ideal method file.

Developed Method File

A method file was developed with the following parameters, as they provided optimal peak separation:

  • 40% to 80% acetonitrile gradient
  • 0.8 mL/min flow rate
  • 25°C temperature
  • Pressure is a gradient, as the mobile phase is a gradient.

HPLC Run Results

The table below depicts the 12 runs conducted, along with the observations and inferences from each:

Chromatograms

The following chromatograms were obtained as a result of our HPLC experiment:

Standard Curve

The standard curve was generated using the Iturin A dilution series, allowing for the quantification of the analyte based on the peak areas from the chromatograms.

The standard curve serves as a reference to determine the concentration of Iturin A in our samples. It correlates the peak area to the concentration of Iturin A in the prepared dilution series.

Standard Curve

HPLC was performed for the following concentrations of pure Iturin A to obtain an ideal standard curve: 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.25 µg/ml, 3.125 µg/ml, and 1.5625 µg/ml. These concentrations were selected to cover a wide range, allowing for accurate quantification and detection of Iturin A in various sample dilutions.

Fig. Cumulative standard curve incorporating the standard curve of each concentration of pure Iturin

Test samples:

LB

Graph showing Iturin peaks in our sample extracted from cells grown in LB

PDB

Fig. Graph showing Iturin peaks in our sample extracted from cells grown in PDB

Blank

Fig. The graph shows the methanol peak, which was used as the blank since both our Iturin A standards and test samples were dissolved in methanol.

The blank helps us understand which peak is methanol and which peak is Iturin, thus helping us identify the right peaks. From the blank we were able to conclude that the initial cluster of peaks that eluted before the dead time were methanol peaks and therefore need not be considered.

Standard Conc. (µg/ml) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Cumulative Area
100 3876505 1532067 1833313 128625 232490 62592 271091 7936683
50 1937832 756545 951640 64932 116880 32414 188259 4048502
25 1074970 418753 517632 35919 64978 17703 207034 2336989
3.125 156370 58850 74599 5251 9038 2752 257202 564062
1.5625 86100 32015 41195 2942 4978 1236 243346 411812

The concentrations of 12.5 and 6.25 µg/ml were not considered due to the following reasons:

  • The 6.25 µg/ml did not yield properly eluted or separated peaks.
  • The volume of the 12.5 µg/ml sample was exhausted in performing multiple HPLC runs to obtain the ideal method file. Therefore, there was not enough volume left to obtain properly eluted and separated peaks.

Baseline errors were subtracted while calculating the area under each peak.

The Standard Curve (Cumulative Area vs Concentration) obtained from the above data is as follows:

Fig. Standard Curve - Cumulative Area vs Concentration

The cumulative area for each test sample was obtained as follows:

Sample Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Cumulative Area
LB 7196 9473 69523 13443 2835 9821 6174 118465

Sample Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Cumulative Area
PDB 14512 35229 82946 67258 14539 44585 9992 269061

Result

The concentration of Iturin A in each sample was calculated using the calibration curve as follows:

LB

y = mx

y = 80453x

118465 = 80453x

x = 118465 / 80453

x = 1.472 µg/ml

The concentration of Iturin A present in the sample extracted from cells grown in LB is 1.472 µg/ml.

PDB

y = mx

y = 80453x

269061 = 80453x

x = 269061 / 80453

x = 3.344 µg/ml

The concentration of Iturin A present in the sample extracted from cells grown in PDB is 3.344 µg/ml.

Comparison of Test Samples with the Standard Samples

1. Black depicts the standard chromatogram.

2. Pink depicts the test sample chromatogram.

LB

PDB

From the above chromatograms, it is seen that the sample extracted from PDB produces higher peaks than the sample extracted from LB. This indicates that it contains a higher concentration of Iturin A, which is reflected in our calculations.

From the above result, we have quantified the amount of Iturin A produced by the natural promoter Pitu.

Further iterations of HPLC will be performed with varying purity levels of extracted Iturin to identify a constant concentration of Iturin produced by our strain (the amount of Iturin produced in different media will be different as demonstrated in our result above).

Notes:

  • In our standard and samples, seven major peaks were identified as opposed to the eight peaks that are supposed to elute for the eight isoforms of Iturin A.
  • For the calculation of Iturin A concentration, the isoforms of Iturin A were not differentiated. The area under every peak was combined to get the final cumulative area as Iturin A is composed of all eight isoforms, whose activities and properties are similar.

References

  1. M. K. Gupta et al., “A comparative review on High-Performance Liquid Chromatography (HPLC), Ultra Performance Liquid Chromatography (UPLC) & High-Performance Thin Layer Chromatography (HPTLC) with current updates,” Current Issues in Pharmacy and Medical Sciences, vol. 35, no. 4, pp. 224–228, Dec. 2022, doi: 10.2478/cipms-2022-0039.
  2. H. Calvo, I. Mendiara, E. Arias, D. Blanco, and M. E. Venturini, “The role of iturin A from B. amyloliquefaciens BUZ-14 in the inhibition of the most common postharvest fruit rots,” Food Microbiol, vol. 82, pp. 62–69, Sep. 2019, doi: 10.1016/j.fm.2019.01.010.
  3. F.-C. Hsieh, T.-C. Lin, M. Meng, and S.-S. Kao, “Comparing Methods for Identifying Bacillus Strains Capable of Producing the Antifungal Lipopeptide Iturin A,” Curr Microbiol, vol. 56, no. 1, pp. 1–5, Jan. 2008, doi: 10.1007/s00284-007-9003-x.
  4. J. Yuan, W. Raza, Q. Huang, and Q. Shen, “Quantification of the antifungal lipopeptide iturin A by high performance liquid chromatography coupled with aqueous two-phase extraction,” Journal of Chromatography B, vol. 879.