Dec 16, 2022

Public workspace Short HPLC gradient method for 20-Hydroxyecdysone (20E) quantification in malaria vectors V.2

DOI
dx.doi.org/10.17504/protocols.io.5qpvoymezg4o/v2
  • 1Tropical Infectious Diseases Research Center (TIDRC), University of Abomey-Calavi, 01 BP 526, Cotonou (Benin);
  • 2Regional Institute of Public Health, University of Abomey-Calavi, BP 384, Ouidah, Benin;
  • 3Department of Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom;
  • 4Wits Research Institute for Malaria, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2000, South Africa.;
  • 5Centre for Emerging, Zoonotic & Parasitic Diseases, National Institute for Communicable Diseases, Johannesburg 2131, South Africa;
  • 6Division of Pharmacology, Department of Pharmacy and Pharmacology, School of Therapeutic Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2000, South Africa
Oswald Yedjinnavenan Djihinto
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DOI: dx.doi.org/10.17504/protocols.io.5qpvoymezg4o/v2
Protocol Citation: Oswald Yedjinnavenan Djihinto, Luc S. Djogbenou, Luisa Nardini, Armorel Van Eyk, Lizette L. Koekemoer 2022. Short HPLC gradient method for 20-Hydroxyecdysone (20E) quantification in malaria vectors. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoymezg4o/v2Version created by Oswald Yedjinnavenan Djihinto
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 09, 2022
Last Modified: December 16, 2022
Protocol Integer ID: 69775
Keywords: 20-hydroxyecdysone (20E), HPLC, Anopheles gambiae, malaria
Funders Acknowledgement:
IUBMB
Grant ID: 2020 Wood Whelan Research Fellowship
Department of Science and Innovation (DSI) and the National Research Foundation (NRF) South African Research Chairs Initiative
Grant ID: UID 64763 to LLK
Abstract
Ecdysteroids are arthropod steroid hormones that are primarily involved in insect moulting. In arthropod vectors, especially in mosquitoes, ecdysteroids of interest include mainly ecdysone (E) and 20-hydroxyecdysone (20E). These two compounds are involved in several important biological processes. Targeting these compounds and their regulatory pathways could lead to the characterisation of novel genetic tools towards implementing new malaria control strategies. To date, there are two main methods for quantifying E and 20E. These methods include an enzymatic method (Enzyme-Linked Immunosorbent Assay (ELISA)) and a chromatographic method (High-Performance Liquid Chromatography (HPLC)). However, for ecdysteroids quantification, the HPLC methods available in literature go from 30 minutes to one hour. Here, we developed a short HPLC gradient method for 20-hydroxyecdysone quantification in the malaria vector Anopheles gambiae. This method was developed specifically when sample material is limited as well as to save time and cost.
Guidelines
  • A new column has to be equilibrated in the mobile phase for at least 1-2 hours before use and any buffer should be removed from the column daily with 20% methanol or Acetonitrile: 80% HPLC grade water and stored in 80% methanol or Acetonitrile: 20% HPLC grade water.
  • The partial loop option should be chosen ("μL-pickup") for sample analysis to inject sample volumes as little as 1μL, resulting in no sample wastage and to avoid large volumes of standard sample preparation.
  • All samples and mobile phase buffers should be filtered through a 0.45 um filter prior to being analysed.
  • The 20E synthesis in mosquitoes is tightly regulated. To quantify the 20E in non-blood feed mosquitoes, be sure that female mosquitoes have sufficient time for mating to occur.
  • All organic mobile phases used for HPLC analysis should be HPLC grade (Sigma Aldridge Inc).
  • All other chemicals and standards should be of the highest purity grade (Sigma Aldridge Inc.)
Materials
  1. Reagents
Reagent20-hydroxyecdysoneSigma AldrichCatalog #H5142-5MG or H5142-10MG
ReagentMethanol HPLCFisher ScientificCatalog #9093-03 ReagentAcetonitrile HPLCfisherCatalog #9012-03 ReagentAcetic acid glacialSigma AldrichCatalog #ARK2183 ReagentWater MilliQ
2. Consumables
  • Eppendorf tubes
  • 2 mL white HPLC glass vials
  • 250 µL conical glass Micro Inserts
  • Membrane Filter 0.45 µm pore size
  • 2 mL syringe
  • Cones tips
  • Micropipettes

3. Equipments
Equipment
Flexar LC
NAME
High-Performance Liquid Chrommatography system
TYPE
Perkin Elmer
BRAND
N2910402
SKU




Safety warnings
Pump pressure should be closely monitored: an increasing pressure toward the limit of the system is an indication of mobile phase leaking (very low pressure) or blockage in the system (very high pressure).
Before start
  • The HPLC system pump should be purged prior to usage to remove air bubbles.
  • The autosampler (if available) should be flushed to clean the injection system.
  • The column should be equilibrated with the mobile phases (starting conditions) for at least 30 min prior to initiating analyses.
Instrument
Instrument
Analytical HPLC separations were performed on the Flexar LC system (Perkin Elmer) with a UV/Vis Detector and a Phenomenex Kinetex RP C18–5 µm column (4.6 x 150 mm).
The detection wavelength was set at 245 nm.
The flow rate was 1 mL/minute.
The mobile phase included gradient elution with Methanol:Acetonitrile (85-15) and 0.5% acetic acid-Water for 16 minutes (See the chromatographic conditions).
The injection volume was 1 µL.
Chromatographic conditions
Chromatographic conditions
Column: Phenomenex, Kinetex® 5 µm C18, 100 Å, 150 x 4.6 mm
Column temp: 20°C
Mobile phase A: 85:15:Methanol:acetonitrile (v:v)
Mobile phase B: 0.5% acetic acid: 95% HPLC grade water (v:v)
Flow rate: 1 mL/min
Detector: UV at 245 nm
Injection volume: 1 µL
Run time: 16 min
Gradient profile: (ramps are linear)
ABCD
% Mobile phases
Time (min) Flow (ml/min) A (MeOH-ACN) B H2O – 0.5% acetic acid
0.5 11090
10 17030
6 11090

Note
If available on the HPLC system, consider choosing the µL-pickup injection mode.

Mobile phase preparation
Mobile phase preparation
Mobile phase A: 85:15 Methanol:acetonitrile (v:v)
Mobile phase B: 0.5% acetic acid: water (v:v)

Preparation of mobile phase A

Mobile phase A was prepared by mixing methanol and acetonitrile to have a ratio of 85:15.
For example, to prepare Amount1 L , combine Amount850 mL of methanol, Amount150 mL of acetonitrile and mix well.

Preparation of mobile phase B

Mobile phase B was prepared by adding glacial acetic acid to water at a ratio of 0.5% glacial acetic acid.
For example, to prepare Amount1 L , combine Amount5 mL of glacial acetic acid and Amount995 mL of MilliQ water and mix.
Note
The solutions preparation may be scaled up as necessary.







Standard samples preparation
Standard samples preparation
The standard samples used were prepared from the 20-hydroxyecdysone (20E) stock solution at (5 µg/µL).
All samples were prepared in methanol to a final volume of 500 µL.
Make three injections for each standard sample.
ABCD
Concentration  (µg/µL) Final volume (µL) Volume of 20E solution (5 µg/µL) in µL Volume of methanol to be added (µL)
2.5 500 250 250
1.25 500 250 250
0.625 500 250 250
0.3125 500 250 250
0.156 500 250 250
0.078 500 7.8 492.2
0.0097 500 0.97 499.03
Linearity, Limit of Detection (LOD) and Limit of Quantification (LOQ)
Linearity, Limit of Detection (LOD) and Limit of Quantification (LOQ)
Example of calculation



Note
In the "SD of intercept" calculation formula, "N" represents the total number of the standard samples.

Total ecdysteroid extraction and 20E quantification
Total ecdysteroid extraction and 20E quantification

Here, female mosquitoes of Anopheles gambiae (COGS strain) were used.

Total ecdysteroid was extracted using a mix of 95% methanol and 10% Ethanol (v:v).

  1. Homogenize 20 adult females (4 days old) in 250 µL of mL of 95% methanol+10% Ethanol in 1.5 mL Eppendorf tubes using plastic pestles.
  2. Vortex and centrifuge for 4 min at 5000 rpm
  3. Transfer the supernatant to another Eppendorf tube
  4. Homogenize the remaining pellet in 250 µL of 95% methanol +10% Ethanol
  5. Vortex and centrifuge for 4 min at 5000 rpm
  6. Poole the supernatant with the first one.
  7. Filter the solution before injection.
HPLC analysis of the extracts

Inject the sample using the same method as with the standard samples.

Note
For the HPLC analysis, it was observed that with the 1 µL injection volume the interpolated X value (the concentration of the sample) falls outside the output range of X for the fitted curve. We then recommend analysing the mosquito extracts with at least 20 µL injection volume and reporting the calculated concentration to 1 µL injection by dividing the calculated value by a factor of 20.