INTRODUCTION:

Calcium sensing receptor (CASR) is a G-protein coupled receptor (GPCR) involved in calcium homeostasis in vertebrates¹. It is primarily expressed in the parathyroid gland and the renal tubules of the kidney. In the parathyroid gland, the calcium sensing receptor controls calcium homeostasis by regulating the release of parathyroid hormone (PTH).

In the kidney it has an inhibitory effect on the reabsorption of calcium, potassium, sodium, and water depending on which segment of the tubule is being activated². Secondary hyperpara thyroidism is an important complication of chronic kidney disease (CKD) and end stage renal disease, particularly among patients receiving dialysis³.

The drugs Cinacalcet and Etelcalcetide are allosteric modifiers of the calcium-sensing receptor and their chemical structure shown in Figure-1. They are classified as a calcimimetics, binding to the calcium-sensing receptor and decreasing parathyroid hormone release⁴. Cinacalcet is chemical named (R)-N-[1-(1-naphthyl)ethyl]-3-[3-(trifluoromethyl) phenyl] propan-1-amine. It is a novel and typical drug used for the treatment of secondary hyperparathyroidism (elevated parathyroid hormone)⁵. Etelcalcetide is a calcimimetic drug for the treatment of secondary hyperparathyroidism in patients undergoing haemodialysis. It is administered intravenously at the end of each dialysis session. Etelcalcetide functions by binding to and activating the calcium sensing receptor in the parathyroid gland⁶.

Figure- 1: Cinacalcet and Etelcalcetide Chemical structures

2-(3-(trifluoromethyl)phenyl)propanal (Figure-2) is one of the probable impurity during synthesis process and shown structural alert for the potential impurity in Drug substance-X and it needs to be control less than the 3.7 ppm in the drug substance. Due to this defined threshold value, the analytical testing limits required for the detection and quantification of impurity is often in the µg/g depending on the dosage of the API. Therefore, gas chromatography (GC) or Liquid Chromatography hyphenated with MS are generally needed to achieve the required specificity and sensitivity.

Figure-2: Chemical Structure of 2-(3-(trifluoromethyl)phenyl)propanal

A very few reports are available in the literature^7-12ondetermination of Cinacalcet and its impurities by HPLC methods, Spectrophotometric estimation of Cinacalcet Hydrochloride in bulk and tablet dosage form were reported¹³, Determination of 3-Trifluoromethyl benzaldehyde impurity in Cinacalcet drug substances was carried out by HPLC⁵,and for the determination of Cinacalcet in plasma were reported^14-15. Forced degradation products of cinacalcet was determined and characterized by using ESI-MS/MS, FT-IR and NMR techniques was carried out¹⁶. Literature survey on Etelcalcetide^17-20 did not reveals analytical methods for the determination of 2-(3-(trifluoromethyl)phenyl)propanal impurity in Etelcalcetide drug. Many trails performed by gas chromatography (GC) and High-performance liquid chromatography (HPLC), there is no fruitful results obtained in recoveries due to matrix effect and therefore, GC or LC hyphenated with mass spectroscopy are generally needed to achieve the required specificity and sensitivity. To the best of our knowledge, no earlier reports have been discussed on trace level determination of 2-(3-(trifluoromethyl)phenyl)propanal impurity by MS. A comprehensive study was taken to develop a method for trace level determination of 2-(3-(trifluoromethyl) phenyl)propanal by GCMS and followed by validation. Some of validations by GCMS for method information referred^21-29.

MATERIAL AND METHODS:

Chemicals and Reagents:

2-(3-(trifluoromethyl)phenyl)propanal impurity was purchased from Symphony Laboratories (Hyderabad, Telangana, India). UPLC/MS grade methanol was obtained from Biosolve, Dieuze, France.

Preparation of Stock Standard Solution and Calibrators:

The dissolving solvent used for the preparation of the standards was methanol. Accurately weighed 50.0mg of 2-(3-(trifluoromethyl)phenyl)propanal impurity standard in 50ml volumetric flask dissolved up to the mark with same solvent, on the basis of the test sample preparation, the latter 2-(3-(trifluoromethyl)phenyl)propanal solution corresponds to 3.7ppm of TAL.

Sample Preparation:

Drug substance test samples were prepared in order to achieve a concentration of 20mg/mL in dissolving solvent.

Instrumentation and Method Conditions:

All GC-MS analyses were performed using an Agilent 7890N GC system (Palo Alto, CA, U.S.A.) hyphenated with an Agilent 5975C inert XL EI/CI MSD with triple axis Mass Spectrometer. An Agilent DB-624 (30m x 0.32mm i.d. x 1.8μm) GC capillary column was used. The oven temperature gradient started at 150°C held for 5 minutes and it was then ramped to 260°C at 20°C/minutes and held for 12 minutes. A 4 mm i.d. ultra inert liner containing glass wool was used. Helium was used as carrier gas with a constant flow rate of 2.0 mL/min. The injector temperature was kept at 180°C in split mode (5:1). The mass detector was operated in electron impact mode (70eV). The source and quadrupole temperatures were set to 230°C and 150°C respectively. Injection volume was 2µL. The MSD transfer line temperature was set at 260°C. Detection was achieved using a single ion monitoring (SIM) mode with a dwell time of 100ms. The data was collected between 2.0 and 10.0 minutes only. The molecular ion at m/z 173 was monitored (Figure-3). Data was acquired and processed using Agilent Mass Hunter software. Method Validation According to our in-house validation guidelines for limit test methods the following validation parameters needed to be evaluated: selectivity, system suitability, Limit of detection (LOD) and Limit of quantitation (LOQ), precision at LOQ, accuracy at LOQ, linearity, accuracy at TAL and at 150% of the TAL and method precision. Briefly, selectivity was assessed by injection of blanks, standards, and test samples, ensuring that no interfering peaks were present at the time of elution of 2-(3-(trifluoromethyl)phenyl)propanal. LOD and LOQ of the method was determined by linearity slope and intercept method. Precision was evaluated by injection of six replicates of sample solutions that were prepared by spiking test samples at LOQ and TAL. Recovery was evaluated by spiking samples with 2-(3-(trifluoromethyl)phenyl) propanal at LOQ (n=3), TAL (n=6) and 150% of TAL (n=3) and comparing the analyte peak area against a pure standard of the same concentration.

Figure-3: 2-(3-(trifluoromethyl)phenyl)propanal impurity EI-Mass spectrum

RESULTS AND DISCUSSION:

Method Validation:

Selectivity is the ability of an analytical method to differentiate the analyte in the presence of other components in a sample. This was demonstrated by analysis of blanks, standard, and test samples (Figure-4 to 7). The sensitivity of the method was demonstrated by determination at the limit of quantitation (LOQ) which was defined by series of standard solution were injected linearity curve was established and by slope and intercept LOD and LOQ values were calculated. The LOQ of the method for 2-(3-(trifluoromethyl)phenyl) propanal was 1.85 ppm. Precision was evaluated by injection of six replicates of sample solutions that were prepared by spiking drug substance test samples at TAL of 3.7 ppm. The calibration curves showed good linearity over the concentration range (LOQ to 150%) of 1.85 to 5.56 ppm. The correlation coefficient was >0.999 (Table-1 and Figure-7). Recovery was evaluated by spiking samples with 2-(3-(trifluoromethyl)phenyl) propanal at LOQ, TAL and 150% of TAL and comparing the analyte peak area against a pure standard of the same concentration. The analyte could be fully recovered (101.1% at LOQ level, 98.6% at TAL and 98.1% at TAL-150%) and shown in table 2 to 4 and Figure-8, no additional matrix effect was observed. In test samples 2-(3-(trifluoromethyl) phenyl)propanal impurity was not detected.

Table 1: System suitability

System suitability	Area
Injection-1	1133.9
Injection-2	1106.16
Injection-3	1103.06
Injection-4	1055.65
Injection-5	1017.6
Injection-6	1004.09
Mean	1070.08
SD	52.50
% of RSD	4.9

Table 2: Linearity

S. No	Concentration (in ppm)	Area
1	1.85	402.62
2	2.78	650.23
3	3.70	925.34
4	4.63	1170.24
5	5.56	1449.78
Correlation Coefficient		0.9998

Figure 7: Linearity graph for 2-(3-(trifluoromethyl)phenyl) propanal

Table 3: Precision and recovery of impurity determination in test sample at LOQ level

Preparation	Area of Standard Solution	Impurity added ppm	Impurity found ppm in Spiked Sample	% Recovery
Prepration-1	394.15	1.85	1.90	102.6
Prepration-2	397.46	1.85	1.91	103.1
Prepration-3	374.69	1.85	1.80	97.4
Prepration-4	365.88	1.85	1.76
Prepration-5	389.23	1.85	1.87
Prepration-6	371.89	1.85	1.79
Average recovery				101.0

Table 4: Precision and recovery of impurity determination in test sample (n=6) at TAL

Preparation	Standard Solution	Impurity added ppm	Impurity found ppm in Spiked Sample	% Recovery
Prepration-1	1186.16	3.70	4.10	110.8
Prepration-2	1176.4	3.70	4.08	110.4
Prepration-3	1043.97	3.70	3.62	97.9
Prepration-4	1054.32	3.70	3.66	98.9
Prepration-5	1039.01	3.70	3.60	97.2
Prepration-6	1037.76	3.70	3.60	97.3
Average recovery				102.1

Figure-8: Recovery graph for 2-(3-(trifluoromethyl)phenyl) propanal at TAL

Table 5: Precision and recovery of impurity determination in test sample (n=3) at 150% of TAL

Preparation	150% Standard Solution area	Impurity added ppm	Impurity found ppm in Spiked Sample	% Recovery
Prepration-1	1356.82	5.56	5.44	97.9
Prepration-2	1351.66	5.56	5.34	96.0
Prepration-3	1310.68	5.56	5.58	100.4
Prepration-4	1329.32	5.56	5.52
Prepration-5	1336.69	5.56	5.57
Prepration-6	1324.32	5.56	5.51
Average recovery				98.1

Table 6: Column flow at 1.8ml/min

System suitability	Area
Injection-1	1233.8
Injection-2	1229.4
Injection-3	1225.6
Injection-4	1258.8
Injection-5	1241.7
Injection-6	1198.1
Mean	1231.23
SD	20.04
% of RSD	1.6

Table 7: Column flow at 2.2ml/min

System suitability	Area
Injection-1	1011.23
Injection-2	1028.2
Injection-3	1019.98
Injection-4	1052.7
Injection-5	1001.54
Injection-6	1033.3
Mean	1024.49
SD	17.94
% of RSD	1.8

Table 8: Column Oven temperature at 145ºC

System suitability	Area
Injection-1	999.54
Injection-2	1002.57
Injection-3	923.59
Injection-4	960.75
Injection-5	983.17
Injection-6	991.22
Mean	976.81
SD	30.06
% of RSD	3.1

Table-9: Column Oven temperature at 155ºC

System suitability	Area
Injection-1	986.42
Injection-2	997.87
Injection-3	960.51
Injection-4	950.36
Injection-5	965.35
Injection-6	970.65
Mean	971.86
SD	17.46
% of RSD	1.8

Figure 4: Blank chromatogram

Figure 5: LOQ Solution Chromatogram

Figure 6: TAL-(3.7 ppm) of impurity solution Chromatogram

Figure 7: Analysis of test sample

Injection	Method Precision	Intermediate Precision
Injection-1	4.10	3.86
Injection-2	4.08	3.94
Injection-3	3.62	4.12
Injection-4	3.66	3.81
Injection-5	3.60	3.65
Injection-6	3.60	3.78
Mean	3.82
SD	0.192563
% of RSD	5.0

4. CONCLUSION:

A GC-MS method for the determination of 2-(3-(trifluoromethyl)phenyl)propanal was developed. Mass spectrometry ensured the method was sufficiently sensitive to control the impurity at trace level. The method was validated and fulfilled our analytical validation criteria for trace level analytical methods. 2-(3-(trifluoromethyl)phenyl)propanal impurity was not detected in test sample.

ACKNOWLEDGEMENTS:

The authors’ special thanks to Mr Nuka Srikanth Kumar and M. Veera Bhadra and the management of Dr.Reddy’s Laboratories Ltd. For permitting to carry out the present work. The authors also wish to thank the colleagues of Analytical Research and Development and Process Research and Development Department for supporting this work. Dr. Reddys communication number for this article: IPDOIPM-00623.

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Received on 08.11.2023 Modified on 07.12.2023

Asian J. Research Chem. 2024; 17(1):25-30.

DOI: 10.52711/0974-4150.2024.00005