Nov 10, 2021
Version 1
Short HPLC gradient method for 20-Hydroxyecdysone (20E) quantification in malaria vectors V.1
- Oswald Yedjinnavenan Djihinto1,
- Luc S. Djogbenou1,2,3,
- Luisa Nardini4,5,
- Armorel Van Eyk6,
- Lizette L. Koekemoer4,5
- 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
- Tropical Infectious Diseases Research Center (TIDRC)
Protocol Citation: Oswald Yedjinnavenan Djihinto, Luc S. Djogbenou, Luisa Nardini, Armorel Van Eyk, Lizette L. Koekemoer 2021. Short HPLC gradient method for 20-Hydroxyecdysone (20E) quantification in malaria vectors. protocols.io https://dx.doi.org/10.17504/protocols.io.by4cpysw
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: October 15, 2021
Last Modified: November 10, 2021
Protocol Integer ID: 54116
Keywords: 20-hydroxyecdysone (20E), HPLC, Anopheles gambiae, malaria
Funders Acknowledgements:
International Union of Biochemistry and Molecular Biology
Grant ID: 2020 Wood Whelan Research Fellowship awarded to OYD.
The authors would like to acknowledge the Department of Science and Innovation (DSI) and the National Research Foundation (NRF) South African Research Chairs Initiative
Grant ID: UID 64763 to LLK, for partial funding
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
- Reagents
20-hydroxyecdysoneSigma AldrichCatalog #H5142-5MG or H5142-10MG
Methanol HPLCFisher ScientificCatalog #9093-03 Acetonitrile HPLCfisherCatalog #9012-03 Acetic acid glacialSigma AldrichCatalog #ARK2183 Water 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
Equipment
Eppendorf Vacufuge Concentrator System
NAME
Eppendorf
BRAND
5301
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 254 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 254 nm
Injection volume: 1 µL
Run time: 16 min
Gradient profile: (ramps are linear)
| A | B | C | D | |
| % Mobile phases | ||||
| Time (min) | Flow (ml/min) | A (MeOH-ACN) | B H2O – 0.5% acetic acid | |
| 0.5 | 1 | 10 | 90 | |
| 10 | 1 | 70 | 30 | |
| 6 | 1 | 10 | 90 |
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 1 L , combine 850 mL of methanol, 150 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 1 L , combine 5 mL of glacial acetic acid and 995 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/L).
All samples were prepared in methanol to a final volume of 500 µL.
Make three injections for each standard sample.
| A | B | C | D | |
| Concentration (µg/L) | Final volume (µL) | Volume of 20E solution (5 µg/L) 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
20-hydroxyecdysone (20E) was detected in total ecdysteroid extract from only female mosquitoes.
Here, female mosquitoes of Anopheles gambiae (COGS strain) were used.
Total ecdysteroid was extracted using methanol according to the method described by McKinney et al., 2017.
CITATION
- homogenize 15 non-blood fed adult females (4 days old) in1 mL of methanol 95% in 1.5 mL Eppendorf tubes using plastic pestles.
- Vortex and centrifuge for 5 min at 13000xg
- Transfer the supernatant to another tube
- Homogenize the remaining pellet in 500 µL of methanol 95% and centrifuge
- Poole the supernatant with the first one.
- Dry the pooled supernatants in a vacuum centrifuge
- Resuspend the dried sample in 500 µL of methanol 100% and 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 5 µL injection volume and reporting the calculated concentration to 1 µL injection by dividing the calculated value by a factor of 5.
Citations
Step 6
McKinney DA, Strand MR, Brown MR. Evaluation of ecdysteroid antisera for a competitive enzyme immunoassay and extraction procedures for the measurement of mosquito ecdysteroids
https://doi.org/10.1016/j.ygcen.2017.08.028