INTRODUCTION:

Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting a substantial portion of the population, primarily those over 50 years old. It is characterized by motor symptoms like bradykinesia, rigidity, tremor, and postural abnormalities, as well as secondary motor symptoms such as gait abnormalities, micrographia, and speech problems. Additionally, PD presents non-motor features like cognitive dysfunction, sleep abnormalities, pain, and autonomic disturbances, impacting patients' overall well-being¹.

The standard treatment for PD is levodopa, which initially provides significant therapeutic benefits. However, its long-term efficacy is often compromised by the emergence of motor complications such as dyskinesias and motor fluctuations². Levodopa also does not effectively address certain symptoms like falls, autonomic dysfunction, and cognitive impairment³.

Safinamide chemically identified as (+)-(S)-2-[[p-[(m-fluorobenzyl)oxy]benzyl]amino] propionamide and marketed under the name XADAGO has emerged as a promising therapeutic agent for PD, with its main effect being the reversible inhibition of the MAO-B enzyme, enhancing dopamine activity and alleviating motor symptoms⁴. Given the potential benefits and unique pharmacological profile of safinamide, it has drawn considerable interest in the field of neurology as a valuable addition to the armamentarium of PD treatments^5-6. As with any active pharmaceutical ingredient (API), ensuring the quality and safety of safinamide mesylate is crucial. The presence of impurities, including genotoxic impurities (GTIs), is a critical consideration in pharmaceutical development to guarantee patient safety. Genotoxic impurities possess the potential to cause genetic mutations or other adverse effects in living organisms, necessitating their strict control and maintenance below acceptable limits^4,7-8. Chemical structures of Safinamide mesylate and N-Nitroso safinamide are shown in Figure 1 and Figure 2.

Figure 1: Safinamide Mesylate

Figure 2: N-Nitroso Safinamide

Therefore, this research work aims to determine and quantify the genotoxic impurity N-Nitroso Safinamide in Safinamide mesylate. Understanding the presence and levels of this impurity is vital for establishing appropriate measures to mitigate potential risks and ensure the well-being of patients receiving safinamide mesylate treatment for Parkinson's disease.

Several research works have been conducted to study Safinamide mesylate and its impurities, aiming to develop reliable and precise analytical methods for its determination and characterization. Liang Zou et al. focused on characterizing process-related impurities and degradation products of safinamide mesylate in bulk drug and developed a stability-indicating HPLC method for their quantification⁹. Vaibhav S Adhao et al. developed an RP-HPLC method to assess the stability of Safinamide mesylate under different stress conditions¹⁰. Pranali C. et al., reported a review of various analytical methods for the selected drug¹¹. Amrutkar G et al., and Vivekkumar K. Redasani et al. each developed HPLC and HPTLC methods for Safinamide determination in bulk and tablet formulations, demonstrating their accuracy and precision^12,13. Ayesha Rehman et al. developed a rapid and sensitive HPLC method to determine safinamide mesylate in bulk and tablet forms¹⁴. Tammisetty et al. established a robust UPLC-MS/MS method for the quantification of safinamide in aqueous solutions and human plasma, offering excellent sensitivity and precision¹⁵. Lastly, Omar M El-Abassy et al. utilized design of experiments (DoE) to optimize an RP-HPLC method for safinamide mesylate and its precursor p-hydroxybenzaldehyde¹⁶, employing green analytical chemistry principles. These diverse research works highlight the importance of accurate and precise methods for determining safinamide mesylate and its impurities, including the focus on N-Nitroso safinamide in the current research.

MATERIALS AND METHODS

Reagent and chemicals

Safinamide mesylate and N-Nitroso safinamide were obtained from BIOPHERE analyticals in Hyderabad. Formic acid, used for liquid chromatography-mass spectrometry (LCMS), was procured from Honeywell in Mumbai, India. LCMS-grade water and methanol were also sourced from Honeywell. Additionally, Milli-Q water (Millipore, USA) was used in the experiments. These carefully selected reagents and chemicals ensured accurate and reliable results throughout the study.

Instrumentation

The HPLC system, an Agilent 1260 Infinity LC coupled with a 6470 TQ detector, served as a robust platform for chromatographic analysis. Additionally, Detector 1, a DAD (Diode Array Detector), and Detector 2, an Ultivo Triple Quadrupole Mass Spectrometer with ESI (Electrospray Ionization), contributed to precise and sensitive detection of analytes.

Data acquisition and analysis were efficiently managed using the Mass Hunter software, ensuring accurate and reliable results. The chromatographic column, a Poroshell HPH-C18 (150 x 4.6mm, 2.7 µm) with temperature control, provided optimal separation and temperature stability during the analyses. Precise measurements were achieved using the Mettler Toledo/XSE205DU analytical balance and the Radwag/MYA 5.4Y micro balance, while the Sartorius micropipette ensured accurate and controlled sample handling. Collectively, these sophisticated and well-maintained instruments and software played a crucial role in ensuring accurate data acquisition and analysis.

Preparation of solutions

Preparation of mobile phase

For the mobile phase, a careful optimization process involving the combination of mobile phase A and mobile phase B was undertaken to achieve the ideal balance of organic and aqueous phases. Mobile phase A was prepared by accurately measuring 1.0 mL of formic acid and diluting it with 1000 mL of water. Thorough mixing was performed to achieve a uniform and consistent solution. Similarly, mobile phase B was prepared by adding 1.0 mL of formic acid to 1000 mL of methanol, and again, ensuring thorough blending to achieve a homogenous mixture.

Preparation of Diluent

The diluent, used as the blank solution in all experiments, was prepared by precisely measuring 10.0 mL of formic acid and adding it to 1000 mL of methanol. Vigorous mixing was performed to achieve complete dissolution and homogeneity of the solution.

Preparation of N-Nitroso Safinamide Standard Stock Solution

To prepare the N-Nitroso Safinamide standard stock solution, approximately 5 mg of the standard was accurately weighed and transferred into a 5 mL volumetric flask. After adding around 3 mL of diluent, the flask was subjected to sonication for 1 minute to facilitate complete dissolution. The solution was then diluted to volume with diluent and thoroughly mixed. Subsequently, the standard stock solution -1 was prepared by transferring 0.36 mL of the N-Nitroso Safinamide standard stock solution into a 20 mL volumetric flask and diluting it to volume with diluent. Careful mixing was performed to ensure proper homogenization.

Preparation of Standard Stock Solution-2

For the preparation of standard stock solution - 2 (18 ppm), 0.5 mL of standard stock solution - 1 was accurately measured and transferred into a 50 mL volumetric flask. Diluent was added to the volume, and the solution was thoroughly mixed to achieve the desired concentration.

Preparation of Standard Solution: (0.18 ppm)

To prepare the standard solution (0.18 ppm), 0.5 mL of standard stock solution - 2 was precisely measured and transferred into a 50 mL volumetric flask. Diluent was added to reach the desired volume, and the solution was thoroughly mixed.

Preparation of sample solution

For the sample solution, approximately 100 mg of the test sample was carefully weighed and transferred into a 10 mL volumetric flask. The sample was dissolved in diluent, and the volume was made up to the mark with diluent. Thorough mixing ensured the uniformity of the solution.

Chromatographic and mass spectrometric conditions

The identification of the genotoxic impurity N-nitroso safinamide in safinamide mesylate required careful selection of chromatographic and mass spectrometric parameters. The Poroshell HPH-C18 column (150 x 4.6mm, 2.7 µm) was chosen for its excellent peak resolution and shape of the target compounds. The 4.6 x 50 mm Ghost Buster column was utilized to prevent interference from remaining chemicals or impurities, ensuring the analysis's reliability. Formic acid and methanol were selected as mobile phases A and B, respectively, to enhance separation efficiency. The specific time/ratio intervals of the mobile phase were meticulously determined through method optimization, achieving optimal separation and resolution of the target analytes. A flow rate of 0.8 mL/min and an injection volume of 10 µL provided efficient elution and sensitivity without sacrificing resolution. The gradient elution method was employed to enhance separation efficiency, particularly for complex samples. The column temperature of 40 °C and sample temperature of 10 °C were carefully controlled to maintain chromatographic system stability and preserve analyte integrity. The AJS ESI ionization mode in positive polarity was chosen for mass spectrometric analysis, ensuring efficient ionization and detection of positively charged ions. The nebulizer pressure (45 psi), nitrogen gas flow rate (350 °C at 12 L/min), and capillary voltage (3500 V) were optimized to enhance ion signal intensity and sensitivity for detecting the genotoxic impurity at low levels.

Method Validation

The developed and optimized method underwent a thorough validation process in strict accordance with the guidelines provided by the International Council for Harmonization (ICH). Precision, specificity, accuracy, linearity, Limit of Detection (LOD), Limit of Quantification (LOQ), range, and robustness were carefully evaluated to ensure the method's reliability and suitability for accurate analysis¹⁷.

Linearity, Precision, Accuracy and Robustness

A series of solutions containing N-Nitroso Safinamide at various concentration levels, ranging from the limit of quantification (LOQ) to 150%, were meticulously prepared to assess linearity. These solutions were injected into the LC-MS/MS system following the specified protocol. For the LOQ standard solution, 0.05 mL of the standard stock solution - 2 was transferred into a 50 mL volumetric flask and diluted with the diluent. Similarly, for the linearity solutions at 50%, 100%, 125%, and 150% levels, appropriate volumes (0.25, 0.5, 0.625, 0.75 mL) of the standard stock solution - 2 were transferred into separate 50 mL volumetric flasks and made up to the volume with the diluent. The linearity graph allowed for the assessment of the correlation between the concentration of N-Nitroso Safinamide and the corresponding response area.

Six different preparations of genotoxic contaminants spiked at a concentration of 0.18 ppm were injected into the chromatographic system equipped with a mass detector to evaluate repeatability (intra-day precision) within the same day and on different days to assess intermediate precision (inter-day precision) and reproducibility.

The accuracy of the proposed method was evaluated through recovery studies, which aimed to determine the closeness of the measured values to the true values. Impurity blend stock solutions were spiked at different concentration levels into the test preparation to assess the recovery of impurities in the presence of the test sample. The accuracy evaluations were conducted at three concentration levels: 50%, 100%, and 150% of the 0.18 ppm level of impurities. Each concentration level was prepared in triplicate using specific volumes (0.25 mL, 0.5 mL, and 0.75 mL) of the standard stock solution. The accuracy test samples were injected into the system, and the results were expressed in terms of standard deviation (SD) and percent relative standard deviation (% RSD) to ensure the reliability and precision of the measurements.

To evaluate the robustness of the method, deliberate adjustments were made to critical parameters, and their influence on the analytical results was thoroughly examined. By comparing the obtained results with the original accuracy conditions, changes in the relative retention time (RRT) value and the percentage level of impurities were determined.

RESULTS AND DISCUSSION

The choice of the Poroshell HPH-C18 column (150 x 4.6 mm, 2.7 µm particle size) played a critical role in achieving exceptional separation efficiency, as it is renowned for providing high resolution and peak shape. Alongside this column, the Ghost Buster column (4.6 x 50 mm) was utilized for efficient sample preparation, further enhancing the method's robustness and reliability. Meticulous attention was given to the chromatographic conditions, including the composition of the mobile phase (formic acid and methanol) and the implementation of gradient elution, which contributed to heightened sensitivity with flow rate 0.8 mL/min (Column temperature 40 °C).

In the realm of mass spectrophotometry, the adoption of the AJS ESI (Electrospray Ionization) ionization mode proved highly advantageous. Operating in the positive polarity, this ionization mode minimized the fragmentation of Safinamide mesylate and its N-Nitroso impurities, preserving their structural integrity. The mass spectrophotometer was operated under specific parameters, including a nebulizer pressure of 45 psi, nitrogen gas flow at 12 L/min and 350 °C, and a capillary voltage of 3500 V. The scan mode employed was Multiple Reaction Monitoring (MRM), known for its high selectivity and sensitivity.

Method Validation

Linearity, Precision, Accuracy and Robustness

The chromatograms of blank, standard, sample, and spiked sample solutions (Figure 3) showed no interferences at the retention times of the impurities, confirming the method's specificity and accurate detection of N-nitroso safinamide without interference from other sample components.

The linearity graph demonstrated a strong linear relationship between the analyte concentration and peak area response across the tested concentration range (LOQ to 150 %, Figure 4). The correlation coefficient was found to be > 0.99 as shown in Figure 5, further confirming the method's effectiveness and accuracy in quantifying impurities. The results and details are presented in Table 3. The LOD concentration was determined to be 0.003 ppm, and the LOQ concentration was found to be 0.009 ppm. The obtained S/N ratios were >3 for LOD and >10 for LOQ as given in Table 4, indicating the method's sensitivity and ability to detect and quantify N-Nitroso Safinamide impurities at low concentrations. Table 5 shows that the method remains stable and reliable even in the presence of variations in flow rate and column temperature.

Table 1: Precision study

Standard solution	System precision	Method precision		Intermediate precision
Standard solution	Area of N-Nitroso Safinamide	Area of N-Nitroso Safinamide	Content of N-Nitroso Safinamide (ppm)	Area of N-Nitroso Safinamide	Content of N-Nitroso Safinamide (ppm)
Inj-1	37288	46962	0.16598	31917	0.18494
Inj-2	41008	49345	0.15821	32554	0.19459
Inj-3	42439	49251	0.17119	33734	0.19274
Inj-4	43741	49484	0.16627	32358	0.19538
Inj-5	44629	52073	0.16960	35601	0.19405
Inj-6	45483	53154	0.16295	35758	0.19611
Average	42431.33	50044.83	0.166	33653.67	0.193
SD	2977.83	2223.45	0.0047	1681.12	0.004
%RSD	7.0	4.4	2.8	4.8	2.1
Bracketing std		45965		39614
Average		49462		34505.1
SD		2549		2725.84
% RSD		5.2		7.9

Table 2: Accuracy study

N-Nitroso Safinamide
Preparation	LOQ	50%	100%	150%
Prep-1	85.92	91.53	90.26	97.18
Prep-2	88.68	92.15	85.97	98.63
Prep-3	89.90	89.95	93.14	98.98
Average	88.2	91.2	89.8	98.3

Table 3: Linearity study of N-Nitroso Safinamide

Level	Conc. (ppm)	Area
LOQ	0.01811	5417
50%	0.09054	28136
100%	0.18107	53454
125%	0.22634	70009
150%	0.27161	83726
Slope	307516.7
Correlation coefficient	0.999

Table 4: LOD and LOQ of Safinamide mesylate

Parameter	Conc. (ppm)	S/N ratio
LOQ	0.01797	15.5
LOD	0.00593	5.1

Table 5: Robustness study of N-Nitroso Safinamide

Prepa ration	Flow rate: 0.88 mL/min	Flow rate: 0.72 mL/min	Column Temp: 45 °C	Column Temp: 35 °C
Prep-1	0.12754	0.16924	0.12523	0.12416
Prep-2	0.14113	0.17217	0.13651	0.13789
Prep-3	0.14262	0.17308	0.14518	0.14359
average	0.137	0.171	0.136	0.135
SD	0.0083	0.0020	0.0100	0.0100
% RSD	6.1	1.2	7.4	7.4
Average	0.16	0.17	0.16	0.16
SD	0.02	0.005	0.02	0.02
% RSD	9.8	2.9	10.4	10.6

Figure 5: Linearity of N-Nitroso Safinamide

CONCLUSION:

A highly sensitive, reliable and precise methodology for the detection of N-Nitroso Safinamide in Safinamide mesylate using ESI-MS/MS analysis was successfully developed. The developed methodology offers valuable insights for quality control and safety assessment in the pharmaceutical industry.

REFERENCES:

1. Lew M. Overview of Parkinson’s disease. Pharmacotherapy. 2007; 27(12): 155S-160S.

2. Thobois S, Delamarre-Damier F, Derkinderen P. Treatment of motor dysfunction in Parkinson’s disease: an overview. Clinical Neurology and Neurosurgery. 2005; 107(4): 269-281.

3. Nagatsu T, Sawada M. L-dopa therapy for Parkinson’s disease: Past, present, and future. Parkinsonism and Related Disorders. 2009; 15: S3-S8.

4. Perez-Lloret S, Rascol O. The safety and efficacy of Safinamide mesylate for the treatment of Parkinson’s disease. Expert Review of Neurotherapeutics. 2016; 16(3): 245–58.

5. Schneider A, Sari AT, Alhaddad H, Sari Y. Overview of therapeutic drugs and methods for the treatment of parkinson’s disease. CNS and Neurological Disorders - Drug Targets. 2020; 19(3): 195–206.

6. Yahr MD. Overview of present-day treatment of parkinson’s disease. Journal of Neural Transmission. 1978; 43(3): 227–38.

7. Reddi A, Mujahid M, Sasikumar M, Muthukrishnan M. A new enantioselective synthesis of the anti-parkinson agent Safinamide. Synthesis. 2014; 46(13): 1751-1756.

8. Malek N, Grosset D. Investigational agents in the treatment of parkinson’s disease: focus on Safinamide. Journal of Experimental Pharmacology. 2012; 5(4): 85-90.

9. Zou L, Sun L, Zhang H, Hui W, Zou Q, Zhu Z. Identification, characterization, and quantification of impurities of Safinamide mesilate: Process-Related Impurities and Degradation Products. Journal of AOAC International. 2017; 100(4): 1029–37.

10. Adhao VS, Thenge RR, Sharma J, Thakare M. Development and validation of stability indicating RP-HPLC method for determination of Safinamide mesylate. Jordan Journal of Pharmaceutical Sciences. 2020; 13(2): 149-159.

11. Baviskar PC, Bachhav RS. An overview on various analytical methods for estimation of Safinamide from its bulk and pharmaceutical dosage form. World Journal of Pharmaceutical Sciences. 2021; 9(11): 114–119.

12. Amrutkar GR, Aher SS, Bachhav RS. Stability indicating RP-HPLC method development and validation for estimation of Safinamide in bulk drug and dosage form. International Journal of Pharmacy and Biological Sciences. 2022; 12(4): 2321–3272.

13. Redasani VK, Mali BJ, Surana SJ. Development and validation of HPTLC method for estimation of Safinamide mesylate in bulk and in tablet dosage form. ISRN Analytical Chemistry. 2012; 2012: 1–4.

14. Rehman A, Nath T, Pasupati T, Harsha K, Sreedhar C. Method for determination of Safinamide mesylate in bulk and tablet dosage form by both RP-HPLC AND HPTLC. Journal of Emerging Technologies and Innovative Research. 2021; 8(8): a421–31.

15. Mohan RT, Balasekhara RC, Srinivasa BP. Application of liquid chromatography with tandem mass spectrometric method for quantification of Safinamide in invitro samples. International Journal of Life Science and Pharma Research. 2020; 10(2): 55–61.

16. El-Sayed HM, Abdellatef HE, Hendawy HAM, El-Abassy OM, Ibrahim H. DoE-enhanced development and validation of eco-friendly RP-HPLC method for analysis of Safinamide and its precursor impurity: QbD approach. Microchemical Journal. 2023; 190:108730.

17. Step. Committee for Medicinal Products for Human Use, ICH guideline M7 on assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk-questions and answers. [cited 2023 Jul 2];

Available from: http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono

Received on 24.12.2023 Modified on 15.02.2024

Research J. Pharm. and Tech 2024; 17(4):1867-1873.

DOI: 10.52711/0974-360X.2024.00296