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Grace.
Sean M. McCarthy, Andrew J. Aubin, and Michael D. Jones
Waters Corporation (Milford, MA, USA)
Application benefit
â– Rapid isolation of gramicidin
â– Load linear response
â– Accurate and high-precision method for analyzing gramicidin
â– It is possible to use other hydrophobic peptides and proteins
Waters Solutions
ACQUITY UPC 2 System
ACQUITY ® SQD
ACQUITY UPC 2 CSH Fluorophenyl Column
EmpowerTM 3 software
Key words
Ultra-high performance convergent chromatography, UPC 2 , hydrophobic peptide, gramicidin
Introduction
The use of reversed-phase liquid chromatography (RPLC) for the analysis of hydrophobic peptides and proteins is difficult because solutions often require the use of detergents to maintain the stability of hydrophobic materials that are prone to aggregation and/or precipitation, seriously affecting them. The recycling, these and other reasons make it difficult to separate hydrophobic peptides and proteins with RPLC.
In this application note, we introduce you to a method for separating a typical transmembrane peptide, gramicidin, using Waters ® ultra-high performance convergent chromatography on the ACQUITY UPC2TM system.
Brevibacterium is a common and well characterized transmembrane peptide material produced by Bacillus, which is used as a topical antibiotic against Gram-positive and certain Gram-negative bacteria, including bacteriocin The composition is formyl-L-valine-glycine-L-alanine-D-leucine-L-alanine-D-valine-L-valine-D-valine-L - a family of tryptophan-D-leucine-LXD-leucine-L-tryptophan-D-leucine-L-tryptophan-aminoethanol, wherein X depends on the gramicidin molecule, That is, Gram A (X = tryptophan), Gram B (X = phenylalanine) and Gram C (X =), which account for about 87.5%, 7.1%, and 5.1% of the total gramicidin, respectively. Tyrosine), 1 requires alternating D and L amino acid units to form a _-helix.
We studied the effects of column chemistry, mobile phase modifiers, and gradient slope on the isolation of gramicidin. The marketed over-the-counter drug (OTC) was isolated using an optimized method to compare the measured gramicidin concentration to the labeled amount. The concentration of gramicidin was determined by mass spectrometry and each material was characterized by selective ion spectroscopy. Using our method on the ACQUITY UPC 2 system, linear and reproducible results were obtained - the measured OTC formulation concentration was 98.4% of the indicated amount.
test
Test Conditions
Unless otherwise stated, the following are the best conditions for all chromatographic final methods.
UPC 2 test conditions
UPC 2 System: ACQUITY UPC 2
Detector: PDA, ACQUITY SQD PDA @ 280nm, resolution 6 nm (compensated 400 to 500 nm)
Column: ACQUITY UPC 2 CSH Fluorophenyl, 3.0 x 100 mm, 1.7 μm
Column temperature: 50 °C
Sample temperature: 15 °C
UPC 2 ABPR: 1885 psi
Injection volume: 1 μL
Flow rate: 2.0 mL/min
Mobile phase A: CO 2
Mobile phase B: methanol with 0.1% TFA (unless otherwise indicated)
Gradient: 20% to 30% B, 1.5min
SQD condition
Ion source: ES+
Taper hole voltage: 20 V
Capillary voltage: 3.7kV
Source temperature: 150 °C
Desolvation gas temperature: 500 °C
Desolvent gas flow rate: 400 L/hr
Cone gas flow rate: 25 L/hr
SIR: 922.6, 930.3, 941.9
Data management
Empower 3 software
sample discription
The gramicidin prepared by Bacillus thuringiensis (Bacillus brevis) was purchased from Sigma Aldrich, and the sample was dissolved in methanol to prepare a solution having a concentration of 0.5 mg/mL, and if necessary, diluted with methanol. Over-the-counter ointments containing gramicidin are purchased from local pharmacies. 0.2 g of ointment was dissolved in 10 mL of n-hexane, then the bacitracin was extracted with 5 mL of methanol, the methanol layer was removed, filtered through a 0.2-μm sintered glass dish, and then directly injected into the ACQUITY UPC 2 system.
Results and discussion
We systematically screened four columns to determine which separation works best. The results are shown in Figure 1. The column screening process can be done very quickly in less than an hour. Under the screening conditions we set, no peaks were detected on the BEH 2-EP and BEH columns. Since other columns showed appropriate chromatographic properties, the non-elution causes of the two were not studied in depth, among them ACQUITY The peak shape of the UPC 2 CSH fluorophenyl column was the best, so the column was used for further research.
Figure 1. Screening of various chemical properties of the column by chromatographic peak shape and retention time of the gramicidin standard. Column specifications for all 3.0x100mm, -2- packed with sub-micron filler; all Mobile phase separation conditions are used A: CO2, mobile phase B, containing the MeOH 0.1% FA, 2 mL / min, 3% B to 25% B , 5min .
In order to isolate the gramicidin material, the effect of the acid modifier was studied. The results showed that a slightly better peak shape was obtained by using trifluoroacetic acid (TFA), which improved the relationship between gramicidin A and gramicidin C. The degree of separation is shown in Figure 2. TFA is known to inhibit mass spectrometry ionization, but the signal for each substance is sufficient to quantify the therapeutic formulation, as will be discussed later. For applications requiring higher sensitivity, it may be necessary to reduce the TFA concentration or use formic acid to achieve the desired detection limit.
Figure 2. Effect of an acidic modifier on the isolation of gramicidin.
After setting the appropriate chromatographic conditions, the separation process was optimized by reducing the gradient time. As shown in Figure 3, we were able to achieve a resolution of 1.4 or higher for each gramicidin component in 1.5 minutes at the same flow rate. Increasing the gradient slope by reducing run time not only achieves efficient separation, but also increases the signal to noise ratio of gramicidin A from 336 to 605.
FIG 3.UV 280-nm trace detection of Separation gramicidin A, B and C.
We tested the optimal separation conditions and were able to detect each substance using single quadrupole mass spectrometry (SQD). Figure 4 shows that each substance was well separated and detected by mass spectrometry, and each gramicidin substance was shown to contain For most M+2H ions, subsequent studies will use these parameters for selective ion monitoring.
Figure 4 : Total ion map -A and adduct ion map -BD for each gramicidin material . The highest intensity ion is selected to assess the content of gramicidin in commercially available antimicrobial formulations. For polypeptide sequences, the red residue is the L -type isomer and the black residue is the D -type isomer.
To assess whether our method is suitable for quantitative analysis of gramicidin in over-the-counter drugs marketed, we used selective ion monitoring on the ACQUITY SQD and the results are shown in Figure 5A. We plot the concentration-peak area curve to get a calibration curve for each substance. As a result, it was found that each component showed a linear response within the test range as shown in Figs. 5B-D. A concentration of each gramicidin substance in the over-the-counter drug was also determined using a calibration curve.
5, FIG. A-25.0, 12.5, 1.25 and standard solution 0.625mg / mL concentration contains superimposed substance selected gramicidin ion spectrum. Panels B , C and D - Linear fit plots of MS peak areas for each of the gramicidin A , B and C.
The developed method was used to assess the concentration and relative abundance of gramicidin material in over-the-counter drugs. As shown in Figure 6, the results of repeated analysis showed that the %RSD value of each gramicidin was small, and the calculated concentration was consistent with the nominal value on the label; we also found the relative abundance of gramicidin and the abundance reported in the literature. Very consistent with 1 .
Figure 6. Superimposed selection of gramicidin A , B and C extracted from antibacterial ointment. The calculated RSD values ​​for the repeated injection analysis (N=3) are within acceptable limits, and the calculated abundance is very high in the literature. Match 1 .
in conclusion
As demonstrated in this application note, the ACQUITY UPC 2 System, in combination with ACQUITYUPC 2 column chemistry, provides a simple, accurate and reproducible analytical method for gramicidin. This work indicates that the ACQUITY UPC 2 system can be used to analyze hydrophobic peptides and may also be used to analyze hydrophobic proteins such as membrane proteins.
references
1. The Merck Index and Encyclopedia of Chemicals, Drugs, and Biologicals. 13th ed. Whitehouse Station, NJ: Merck Research Laboratories; 2001.
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