RP-HPLC Method for Quantification of Etelcalcetide in Bulk and Parentral Dosage Form

 

Krishna Kishore Adireddy*, Srinivasa Rao Baratam, Nagarjuna Hari Pratap S.

Department of Pharmaceutical Analysis, St. Ann's College of Pharmacy, Vizianagaram,

Andhra Pradesh-535003, India.

 

ABSTRACT:

A simple, rapid, accurate and precise RP-HPLC method was developed and validated for the determination of Etelcalcetide in bulk and parentral dosage form. Quantification of the drug was achieved on Shimadzu HPLC comprising of LC- 20 AD binary gradient pump, a variable wavelength programmable SPD-20A detector and SCL system controller. C18G column (250 mm x 4.6 mm, 5 μ) as stationary phase with mobile phase consisting of acetonitrile: methanol :water in the ratio of 25: 45 :30 v/v. The method showed a good linear response in the concentration range of 3.75-22.5 μg/ml with correlation coefficient of 0.9999. The flow rate was maintained at 1.0 ml/min and effluents were monitored at 238 nm. The retention time of etelcalcetide was 6.201 min. The method was statistically validated for accuracy, precision, linearity, ruggedness, robustness, solution stability, selectivity and sensitivity. The results obtained in the study were within the limits of ICH guidelines and hence this method can be used for the determination of etelcalcetide in bulk and parentral dosage form.

 

 

INTRODUCTION:

Increased cardiovascular-related mortality is commonly observed in chronic kidney disease (CKD). The increased burden of cardiovascular disease is, in part, due to the development of vascular calcification. Elevated levels of parathyroid hormone (PTH) and phosphorus in CKD patients are critical for the initiation and progression of vascular calcification in CKD. While increased serum calcium is not common in untreated CKD patients with secondary hyperparathyroidism (SHPT), it may be elevated by certain treatment regimens further contributing to the development of vascular calcification.

 

Etelcalcetide chemically, (2R)-3-[[(2S)-2-acetamido-3-[[(2R)-1-[[(2R)-1-[[(2R)-1-[[(2R)-1-[[(2R)-1-[[(2R)-1-amino-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-1-oxopropan-2yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino] -1-oxopropan-2-yl]amino]-3-oxopropyl]disulfanyl]-2-aminopropanoic acid having molecular formula C₃₈H₇₃N₂₁O₁₀S₂ and molecular weight 1048.26 g/mol. Etelcalcetide is a calcimimetic¹ and calcium-sensing receptor (CaSR) agonist composed of a synthetic peptide comprised of seven D-amino acids that can be used to treat secondary hyperparathyroidism (sHPT) in hemodialysis patients with chronic kidney disease (CKD)^2-9. It is administered intravenously at the end of each dialysis session. Upon intravenous administration, etelcalcetide mimics calcium and allosterically binds to and activates the CaSR expressed by the parathyroid gland. This suppresses the synthesis and secretion of parathyroid hormone (PTH), thereby reducing PTH levels and lowering serum calcium and phosphorus levels. Elevated PTH is often observed in patients with CKD and is associated with dysregulated calcium-phosphate homeostasis¹⁰. It is available under the trade name Parsabiv¹¹ is currently owned by Amgen and Ono Pharmaceuticals in Japan. The spectrophotophotometric methods have a lack of selectivity and sensitivity, so chromatorgraphic method is a method of choice for estimation of etelcalcetide. Out of many chromatorgraphic methods, HPLC^12-21 is a versatile analytical technique with good sensitivity and selectivity. To best of our knowledge there is only one RP-HPLC²² method reported for estimation in dosage form, so there is a need of more RP-HPLC methods required. Thus, the present investigation aimed to develop a suitable, simple, precise, accurate, robust, and reproducible RP-HPLC method for the quantification of etelcalcetide in bulk and parentral dosage form.

 

Fig. 1: Structure of Etelcalcetide

 

MATERIALS AND METHODS:

Instrumentation:

 Chromatographic separation was performed on a Shimadzu LC-20AD HPLC system equipped with a C18 G column (250 × 4.6 mm i.d, 5 μm particle), binary pumps, degasser, variable wave length detector and Rheodyne injector with 20μl loop volume. ‘LC solution’ software was used to collect and process the data.

 

Chemicals and Reagents:

Etelcalcetide(ECD) pure drug (purity > 99.00 %) was purchased online. Marketed formulation PARSABIV® injection (Ono Pharmaceutical Co., Ltd) labeled to contain 5mg/ml of etelcalcetide procured online. HPLC grade water and acetonitrile was from MERCK India Ltd. HPLC grade methanol was from standard reagent Pvt Ltd Hyderabad. Nylon membrane filters 0.2µm and 0.45µm were from Pall Life Sciences Mumbai, India.

 

Chromatographic conditions:

The chromatographic system used for the method development and validation consisted of Shimadzu HPLC comprising of LC-20AD binary gradient pump, a variable wavelength programmable SPD-20A detector and an SCL 20A system controller. A Rheodyne injector 7725i fitted with a 20μL loop was used and data were recorded and evaluated by use of LC solutions software version 5.0. Separation was performed on a Enable C18G (250 × 4.6 mm i.d.,5µ) at ambient temperature. A mixture of acetonitrile: methanol: water in the ratio of 25:45: 30 v/v was found to be the most suitable mobile phase for ideal chromatographic analysis of etelcalcetide. The solvent mixture was filtered through 0.45μ membrane filter and sonicated before use. It was pumped through the column at a flow rate of 1.0mL/min. Injection volume was 20μL and the column was maintained at room temperature . The column was equilibrated by pumping the mobile phase through the column for at least 30 minutes prior to the injection of the drug solution. The detection of the drug was monitored at 238nm. The run time was set at 10 min. The optimized chromatographic conditions are shown in Table 1.

 

Table 1: Optimized chromatographic conditions

Parameters	Conditions
Stationary Phase(Column)	C18 (250 × 4.6 mm i.d.,5µ)
Mobile Phase	Acetonitrile: Methanol:Water (25:45:30,v/v)
Flow rate(ml/min)	1.0 mL/min
Run time(min)	10 min
Column temperature (°C)	Ambient
Volume of injection loop(μL)	20
Detection wavelength(nm)	238 nm
Retention time(min)	6.201

 

Fig. 2: Chromatogram of standard solution

 

Preparation of mobile phase:

Mobile phase was prepared by mixing 250mL of HPLC grade acetonitrile, 450mL methanol with 300ml of HPLC grade water. The mobile phase was sonicated for 10 min and filtered through the 0.45μm membrane filter.

 

Preparation of standard stock solutions:

The standard stock solutions of 1000μg/mL of the etelcalcetide were prepared by dissolving 50mg of pure etelcalcetide in the mobile phase in a 50mL volumetric flask and the volume was made up to the mark. Resulting solutions were further diluted with mobile phase to obtain a final concentration of 100μg/mL and stored under refrigeration. Aliquots of standard stock solutions were put in a 10mL volumetric flask and diluted up to the mark with mobile phase. In such a way, the final concentrations of the etelcacetide were in the range of 3.75–22.5 μg/mL.

Preparation of sample solution:

To determine the content of etelcalticide in intravenous injetion (Label claim: 5mg/mL) the whole content of one vile was removed and then mixed thoroughly. The volume equivalent to 5mg of etelcalcetide was taken and dissolved in 50ml of methanol. The resulting solution (5ml) was transferred to a 10ml volumetric flask and diluted up to the mark with mobile phase. The final solution was filtered through 0.45μ membrane filter using injection filter. A 20µL of the filtrate was injected into chromatographic system. The peak area of the etelcalcetide was determined and concentration was found using linear regression equation obtained from calibration curve.

 

Method validation:

The developed method was validated as per ICH guidelines²³ by evaluating linearity, accuracy, precision, robustness, ruggedness, detection limit, quantification limit and stability. Coefficients of variation and relative errors of less than 2% were considered acceptable.

 

System Suitability Test:

Before performing validation experiments, system suitability test (SST) has to be applied to indicate that HPLC system and method are capable of providing data with admissible quality. SST was performed by investigating capacity factor, tailing factor, theoretical plates number and also relative standard deviation (RSD) of the peak areas.

 

Stability:

Stability was assessed by analyzing QC standard solutions after keeping them at room temperature for 72 h. Obtained results were investigated as recovery values and compared to the freshly prepared solutions.

 

Linearity:

A stock solution of etelcalcetide of 1000 μg/mL was prepared with mobile phase. From it, various working standard solutions were prepared in the range of 3.75 to 22.5 μg/ml and injected into HPLC. It was shown that the selected drug had linearity in the range of 3.75–22.5 μg/ml. The calibration plot (peak area of etelcalcetide versus etelcalcetide concentration) was generated by replicate analysis (n=9) at all concentration levels and the linear relationship was evaluated using the least square method within Microsoft Excel® program.

 

Accuracy:

The accuracy of the method was carried out using one set of different standard addition methods at different concentration levels, 50%, 100% and 150%, and then comparing the difference between the spiked value (theoretical value) and actual found value.

 

Precision:

The precision of the method was ascertained from the peak area obtained by actual determination of six replicates of a fixed amount of the drug (15μg/ml). The precision of the assay was also determined in terms of intra- and inter-day variation in the peak areas of a set of drug solutions on three different days. The intra- and inter-day variation in the peak area of the drug solution was calculated in terms of relative standard deviation (RSD).

 

Robustness:

Robustness of the proposed method for was carried out by the slight variation in flow rate, analytical wavelength and mobile phase ratio. The percentage recovery and RSD were noted for etelcalcetide .

 

Ruggedness:

The test solutions were prepared as per test method and injected under variable conditions. Ruggedness of the method was studied by different analysts.

 

Detection limit and quantification limit:

The limit of detection (LOD) and limit of quantification (LOQ) were established based on the calibration curve parameters, according to the following formulas:

LOD=3.3SD/slope

LOQ=10SD/slope

 

or detection limit=3.3σ/s, quantification limit=10σ/s, where σ is the standard deviation of y-intercept of regression line, and s is the slope of the calibration curve.

 

Specificity:

Specificity was measured by analysing the standard solutions in the presence of an excipient (dextrose) to determine any interference in the percentage recovery. Standard solutions containing 10 mg of each drug were spiked with 50% (5mg), 100% (10mg) or 150% (15mg) of dextrose and analyzed for etelcacitide(ECD) recovery by HPLC. The acceptable level of interference was <0.5.

 

RESULTS AND DISCUSSION:

Method validation:

System Suitability Test:

After setting the optimum conditions, system suitability parameters for the developed method were determined and compared with recommended limits. To determine the parameters, the study was performed with standard solution of 15µg/ml concentration and the results were acquired from six injections. System suitability parameters of the method were demonstrated in Table 1. According to results, all of the system suitability parameters were within the recommended limits and the method was found to be suitable for the analysis.

 

Table 1: Results of system suitability test                             (n = 6)

Parameter	Criteria	Result
Capacity factor(k’)	k’> 2	6.824
Tailing factor (T)	T < 2	1.426
Theoretical plates (N)	N> 2000	2911
% RSD (peak area)	% RSD ≤ 1	1.13

 

Stability:

The sample solution stability was analyzed by injecting the same solution at 0, 12, 24, 48 and 72 h. Identical change was not observed in the developed method. Also, results were found within acceptable limits (RSD < 2 ), which are summarized in Table 2.

 

Table 2: Stability data of Etelcalcetide(standard solutions)

Time (hr)	Assay(%)	% Difference
Initial	100.12	----
After 12 hr	100.02	0.09
After 24 hr	99.73	0.38
After 36 hr	99.21	0.91
After 48 hr	98.98	1.13
After 72 hr	98.31	1.80

 

Linearity and sensitivity:

Linearity study was performed with calibration standards with 3.75, 7.5, 11.25, 15, 18.75 and 22.5 µg/ml concentrations. The standards were injected in triplicate. Calibration curves were obtained by plotting the peak areas against the given concentrations. The calibration curve was evaluated by the determination coefficient. The determination coefficient (R²) of the calibration curves was 0.9999. Therefore, the calibration curve for etelcalcetide was found to be linear within the range of 3.75-22.5 µg/ml concentrations. The regression equations were calculated from the calibration graphs. The sensitivity of the analytical method was evaluated by determining the limits of detection (LOD) and quantitation (LOQ). The values of LOD and LOQ are given in Table 3. The low values of LOD and LOQ indicates the sensitivity of method.

Table 3: Spectral and statistical data for determination of Etelcalcetide by proposed RP-HPLC method.

Parameter	Result
Detection wavelength (nm)	238
Linearity range (µg/ml)	3.75-22.5
Coefficient of determination (r2)	0.9999
Regression equation (Ya)	Y= 21732x+768.67
Slope (m)	21732
Intercept (c)	768.67
Limit of detection, LOD (µg/ml)	0.09
Limit of quantitation, LOQ (µg/ml)	0.27

^aY = mx + c, where x is the concentration (µg/ml).

 

Fig. 3: Linearity graph of Etelcalcetide

 

Accuracy:

To study the reliability, the suitability, and the accuracy of the method, recovery experiments were carried out. Known quantities of the pure drug were added to the preanalyzed sample to make samples at the levels of 50 %, 100 %, and 150 %, and were assayed by the proposed method. Accuracy was calculated as the percentage of recovery. For the accuracy parameter, values greater than 99.2% recovery were acquired for the three concentration levels tested (50, 100, and 150%), as demonstrated in Table 4. Therefore, the method was considered accurate.

 

Table 4: Accuracy results

% Level	Concentration (µg/mL)		Recovery(%)	Statistical Results
	Formulation	Pure drug		Mean	SD	%RSD
50	15	7.5	99.4
50	15	7.5	99.1	99.3	1.21	1.22
50	15	7.5	99.5
100	15	15	100.1
100	15	15	100.6	100.03	1.72	1.72
100	15	15	99.4
150	15	22.5	99.2
150	15	22.5	99.7	99.6	0.87	0.88
150	15	22.5	100.1

 

Precision:

The precision was demonstrated at three levels: repeatability, intermediate precision, and reproducibility (between laboratories’ precision). Each level of precision was investigated by 3 sequential replicate of injections of three concentrations of 7.5, 15 and 22.5 µg/mL. The precision was expressed as relative standard deviation (RSD) or coefficient of variation (CV). The results of three levels of precision are shown in Table 5.In all instances the %RSD value was less than 2 indicates the precision of the developed method.

 

Table 5: Precision results

Precision	Results
	Concentration (µg/mL)	% RSD of Peak area	% RSD of Retention Time
Repeatability	7.5	0.67	0.08
	15	0.81	0.06
	22.5	1.66	0.25
Intermediate precision	7.5	1.88	0.07
	15	0.31	0.09
	22.5	0.88	0.03
Reproducibility	7.5	1.61	0.29
	15	1.11	0.13
	22.5	0.23	0.08

 

 

Robustness and ruggedness

Robustness of the method was studied by deliberate variations of the analytical parameters such as flow rate (1.0±0.1 mL/min), mobile phase composition (± 5% organic phase) and analytical wavelength (±2 nm). The results are given in Tables 6. The slight deliberate variation in analytical parameters doesn’t affected the results and % RSD value was found to be less than 2% indicates the robustness of the method. Ruggedness of the method was carried out by different analysts. The results are displayed in Table 7.The analytical results of the two analyst are similar and % RSD values were less than 2% indicates the rugged method.

Table 6: Robustness results

Parameter	Variation	Observed value
		% RSD of area	% RSD of R.T	Tailing factor(T)	Theroteical plates(N)
Flow rate	0.9	0.21	0.08	1.41	2912
	1.1	0.53	0.09	1.42	2925
M.Phase Composition	30 % acetonitrile	0.64	0.08	1.42	3087
	20 % acetonitrile	0.62	0.13	1.42	2902
Wavelength	240 nm	0.85	0.07	1.42	2921
	236 nm	0.89	0.06	1.41	2907

 

Table 7: Ruggedness results

Analyst	Observed value
	% RSD of area	% RSD of R.T	Tailing factor(T)	Theroteical plates(N)
Analyst I	0.65	0.05	1.42	2921
Analyst II	0.84	0.09	1.42	2943

 

Mobile phase stability:

The stability of the mobile phase was evaluated, so the mobile phase was stored at 4–8 °C for 1 week. The aged mobile phase was compared using a freshly prepared one. The mobile phase was stable up to 1 week at              4–8 °C.

 

Specificity

There is no interference from the added excipient at the retention time of etelcalcetide (ECD) as shown in Fig 4 indicates the specificity of the method.

 

Fig. 4: Specificty graph

 

Sample Analysis

The developed and validated method was applied for analysis of intravenous injection contain etelcalcetide. The sample was analyzed in triplicate. The amount of etelcalcetide in the samples was calculated from calibration curve equation and recovery and SD values were determined. The results of analysis are given in Table 8. The recoveries were in good agreement with the label claims. The chromatogram obtained were clear as shown in Fig. 5. It was concluded that the method can be applied successfully for the analysis of etelcalcetide in parentral preperation.

 

Fig. 5: Chromatogram of sample solution of Etelcalcetide

 

Table 8: Recovery results of formulation

Sample	Labelled amount (mg/ml)	Amount obtained* (mg/ml)	Percentage Recovery* ±SD
PARSABIV® injection (Etelcalcetide)	5 mg/ml	4.92	98.42 ± 0.64

* Average of five determinations

 

CONCLUSION:

The developed RP–HPLC method was found to be suitable for the analysis of etelcalcetide in bulk form and in perentral preperations, and was found to be simple, reliable, sensitive, economical. The parameters established in the validation demonstrated that the method is selective, linear, precise, accurate, free of matrix effects, and it is robust.

 

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Accepted on 17.11.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(10):5521-5526.