METHOD DEVELOPMENT
6. METHOD DEVELOPMENT
Materials and Instruments:
The following are the list of instruments/Equipments, chemicals/reagents and standards to
perform the HPLC Analysis of the drug Etelcalcetide.
Equipments:
Table-5.1: List of Equipments
[Link]. Instruments/Equipments/Apparatus
1. HPLC WATERS with Empower2 Software with Isocratic with UV-Visible
Detector.
2. T60-LABINDIA UV – Vis spectrophotometer
3. High Precision Electronic Balance
4. Ultra Sonicator (Wensar wuc-2L)
5. Thermal Oven
6. Symmetry C18 Column, 250 mm x 4.6 mm and 5µm particle size
7. PH Analyser (ELICO)
8. Vaccum Filtration Kit (Labindia)
Chemicals and Reagents:
Table-5.2: List of Chemicals used
[Link]. Name Grade Manufacturer/Supplier
1. HPLC grade water HPLC Sd fine-Chem ltd; Mumbai
2. Methanol HPLC Loba Chem; Mumbai.
3. Ethanol A.R. Sd fine-Chem ltd; Mumbai
Department of Pharmaceutical Analysis Page 1
METHOD DEVELOPMENT
4. Acetonitrile HPLC Loba Chem; Mumbai.
5. DMSO A.R. Sd fine-Chem ltd; Mumbai
6. DMF A.R. Sd fine-Chem ltd; Mumbai
Working Standard: Working Standard of Etelcalcetide: 10ppm
Solubility Study:
Table-5.3: Solubility Results
Solvents Solubility
Methanol Soluble
Ethanol Soluble
Acetonitrile Soluble
DMSO Soluble
Dichloromethane Soluble
Chloroform Soluble
Water Practically Insoluble in
Water
Hydrochloric acid Sparingly Soluble
HPLC Instrumentation & Conditions: The HPLC system employed was HPLC WATERS
with Empower2 Software with Isocratic with UV-Visible Detector.
Standard Preparation for UV-Spectrophotometer Analysis:
The Standard Stock Solutions – 10 mg of Etelcalcetide standard was transferred into 10 ml
volumetric flask, dissolved & make up to volume with Methanol. Further dilutions were done
Department of Pharmaceutical Analysis Page 2
METHOD DEVELOPMENT
by transferring 1 ml of the above solution into a 10ml volumetric flask and make up to volume
with methanol to get 10ppm concentration.
It scanned in the UV spectrum in the range of 200 to 400nm. This has been performed to know
the maxima of Etelcalcetide, so that the same wave number can be utilized in HPLC UV detector
for estimating the Etelcalcetide.
Fig-: Double Beam UV Spectrophotometer
Fig-: UV-Spectrum for Etelcalcetide
Observation: While scanning the Etelcalcetide solution we observed the maxima at 245nm.
DIFFERENT TRIALS FOR CHROMATOGRAPHIC CONDITIONS
Department of Pharmaceutical Analysis Page 3
METHOD DEVELOPMENT
Table-: Different Chromatographic Conditions
Column Used Mobile Phase Flow Wave Observatio Result
Rate length n
Zorbax C18, 250 mm Methanol : 0.9 245nm Extra peaks Method
x 4.6 mm and 5µm Acetonitrile = 30 : 70 ml/min rejected
Column
Phenomenex C18, 250 Methanol : 1.0 245nm Stabilizatio Method
mm x 4.6 mm and Acetonitrile = 60 : 40 ml/min n was not rejected
5µm Column Good
Symmetry C18, 250 Methanol : 1.0 245nm Improper Method
mm x 4.6 mm and Acetonitrile ml/min peak rejected
5µm Column
= 50 : 50 separation
Symmetry C18, 250 Methanol : Phosphate 1.0 245nm Tailing Method
mm x 4.6 mm and Buffer (0.01M) (pH- ml/min peaks rejected
5µm Column 2.8)
= 40 : 60
Symmetry C18, 250 Methanol : Phosphate 1.0 245nm Tailing Method
mm x 4.6 mm and Buffer (0.02M) (pH- ml/min peaks rejected
5µm Column 3.2)
= 60 : 40
Symmetry C18, 250 Methanol : Phosphate 1.0 245nm Proper Method
mm x 4.6 mm and Buffer ml/min Peak Accepted
5µm Column
(0.02M) (pH-3.8)
= 70 : 30
Department of Pharmaceutical Analysis Page 4
METHOD DEVELOPMENT
Fig-: Chromatogram for Trial-1
Table-: Results of Chromatogram for Trial-1
[Link]. Peak Name Rt Peak Area Height USP Tailing USP Plate Count
1 Etelcalcetide 2.970 45867 2546 2.18 2168
Fig-: Chromatogram for Trial-2
Table-: Results of Chromatogram for Trial-2
[Link]. Peak Name Rt Peak Area Height USP Tailing USP Plate Count
1 Etelcalcetide 3.539 45867 2546 2.18 2168
Department of Pharmaceutical Analysis Page 5
METHOD DEVELOPMENT
Fig- 5.7: Chromatogram for Trial-3
Table-5.8: Results of Chromatogram Trial-3
[Link]. Peak Name Rt Peak Area Height USP Tailing USP Plate Count
1 Etelcalcetide 4.660 685975 42548 1.57 3659
Fig- 5.7: Chromatogram for Trial-4
Table-5.8: Results of Chromatogram Trial-4
[Link]. Peak Name Rt Peak Area Height USP Tailing USP Plate Count
1 Etelcalcetide 2.497 685975 42548 1.57 3659
Department of Pharmaceutical Analysis Page 6
METHOD DEVELOPMENT
Fig- 5.7: Chromatogram for Trial-5
Table-5.8: Results of Chromatogram Trial-5
[Link]. Peak Name Rt Peak Area Height USP Tailing USP Plate Count
1 Etelcalcetide 2.720 685975 42548 1.57 3659
Fig-: Chromatogram for Trial-6
Table-: Results of Chromatogram Trial-6
[Link]. Peak Name Rt Peak Area Height USP Tailing USP Plate Count
1 Etelcalcetide 2.775 715268 47844 1.35 5857
Department of Pharmaceutical Analysis Page 7
METHOD DEVELOPMENT
Optimized Chromatographic Conditions:
Column : Symmetry C18, 250 mm x 4.6 mm i.d.5µm particle size
Mobile Phase : Methanol: Phosphate Buffer (0.02M) (pH-3.8) (70: 30% v/v)
Flow Rate : 1.0ml/minute
Wave length : 245 nm
Injection volume : 10 µl
Run time : 7 minutes
Column temperature : Ambient
Fig-: Chromatogram for Blank Solution
Fig-: Optimized Chromatogram for Etelcalcetide
Department of Pharmaceutical Analysis Page 8
METHOD DEVELOPMENT
Table-: Results of Optimized Chromatogram
[Link]. Peak Name Rt Peak Area Height USP Tailing USP Plate Count
1 Etelcalcetide 2.775 715268 47844 1.35 5857
Preparation of 0.02M Phosphate Buffer (pH-3.8): Prepare 800 mL of distilled water in a
suitable container. Add 2.72172g of Potassium dihydrogen Phosphate to the solution to the
solution. Adjust solution to final desired pH 3.8 using diluted solution of orthophosphoric acid
and add distilled water until volume is 1 Litre.
Preparation of Mobile Phase: Mix a mixture of 0.02M Phosphate Buffer (pH-3.8) 700 ml
(70%) and 300 ml Methanol HPLC (30%) and degas in ultrasonic water bath for 15 minutes.
Filter through 4.5 µ filter under vacuum filtration.
Preparation of Standard Solution:
Accurately weigh and transfer 10 mg of Etelcalcetide working standard into a 10ml of clean dry
volumetric flasks add about 7ml of Diluents and sonicate to dissolve it completely and make
volume up to the mark with the same solvent. (Stock solution)
Further pipette out 0.1ml of Etelcalcetide from the above stock solutions into a 10ml volumetric
flask and dilute up to the mark with Diluent.
Result: The selected and optimized mobile phase was Methanol: Phosphate Buffer (70: 30%
v/v) and conditions optimized were flow rate (1.0 ml/minute), wavelength (245nm), Run time
was 07 mins. Here the peak has shown better theoretical plate count and symmetry. The
proposed chromatographic conditions were found appropriate for the quantitative determination
of the Etelcalcetide drug.
Department of Pharmaceutical Analysis Page 9