Development and Validation of a Rapid and Sensitive LC-APCI-MS/MS Method for the determination of three N-nitrosamine impurities in Varenicline tartrate drug substance and drug products

Uppalapati Vidyamani¹*, Dittakavi Ramachandran², Mannem Durga Babu³,

Sagi Samba Murthy Raju⁴, Korrapati Uma Maheswar⁵

^1,2Department of Chemistry, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India.

³Department of Chemistry, SVRM College, Nagaram, Guntur, Andhra Pradesh, India.

⁴Department of Chemistry, KVR, KVR and MKR College, Guntur, Andhra Pradesh, India.

⁵Department of Chemistry, Bapatla Engineering College, Guntur, Andhra Pradesh, India.

*Corresponding Author E-mail: uvidyamani@gmail.com

ABSTRACT:

Nitrosamines impurities may be present in low levels in a variety of products that people are exposed to every day. After thorough investigations, regulatory agencies have outlined that the formation of nitrosamines is possible in the presence of secondary, tertiary, or quaternary amines and nitrite salts under acidic reaction conditions. For safety purpose, all API manufacturers must be developed methods to monitor the low levels of each nitrosamine impurity before the release the individual batches. In this article, we have developed novel, rapid and sensitive liquid chromatography Atmospiric pressure chemical ionization Mass spectroscopy (LC-APCI-MS/MS) method for the determination of N-Nitroso Dimethylamine (NDMA), N-Nitroso Diethylamine (NDEA) and N-Nitroso varenicline impurities in the Varenicline Tartrate drug substance and Drug products. Chromatographic separation is achieved using Inert sustain AQ C18 150 × 4.6mm, 3.0μ column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at the 0.6 mL/min flow rate by using gradient mode of elution upto the 25 minutes total run time. The three impurities are successfully ionized and quantified in positive mode of atmospheric pressure chemical ionization (APCI) using multiple reaction monitoring (MRM). This method was validated according to ICH Q2 (R1) guidelines. The method provided excellent S/N ratios with a high linearity range of 0.66-19.88 ppm with respect to Varenicline Tartrate sample concentrations with a regression coefficient >0.995. The recovery for the method was established with a protocol of three-step sample preparation and was satisfactory within 85-115%. The determined LOQ (0.66 ppm) and LOD (0.22 ppm) values are very low with respect to sample concentration which shows the sensitivity Performance of the method. The method can be routinely applied for the detection of N-nitrosamine impurities (NDMA, NDEA and N-Nitroso varenicline) in Varenicline Tartrate drug substance and Drug products.

KEYWORDS: Nitrosamine impurities, Varenicline tartrate, LC-APCI-MS/MS, Development and validation.

INTRODUCTION: 

N-Nitrosamines are known potential human carcinogens which are produced during the reaction of a nitrosation agent (such as sodium nitrite) with a primary or secondary amine (such as dimethylainmine).

Their formation can occur only under certain conditions, including strongly acidic conditions¹. Under acidic conditions, sodium nitrite converts to nitrosonium cation which can further react with an amine to produce a nitrosamine. N-Nitrosamines are known to generate in some sartan manufacturing processes during the tetrazole ring formation step through the reaction of dimethylantine² (or any other potential amines which may be present as an impurity in other process solvents such as Dimethylformamide (DMF), Dimethylacetamide (DMAC), N-Methylpyrrolidone (NMP), Triethylamine (TEA) and Tetra butyl ammonium bromide (TATB) with sodium nitrite under acidic conditions (formation of nitrous acid). Sodium nitrile is commonly used to quench the unreacted sodium azide (NaN3) used in the tetrazole formation. Therefore, for a nitrosamine to form during the tetrazole ring formation step, the nitrous acid generated from sodium nitrite during the azide quenching step must come into contact with a primary or secondary amine that may be present as an impurity in the process solvent³. FDA was investigated the presence of nitrosamine in certain drug products. From 2018 several drug products (ARBs, ranitidine, nizatidine, and metformin) have been found to contain unacceptable levels of nitrosamines. These impurities are formed by the synthesis process of API’s, degradation studies, storage conditions and excipient interactions or contaminations^4-9. USFDA and other regulatory agencies have published guidelines for the control of nitrosamine impurities in drug substances and drug products¹⁰. As per USFDA and EMA guidelines, preliminary evaluation for the presence of possible nitrosamine impurities in non Sartans drug substance to be evaluated. Varenicline tartrate fall under this classification, therefore the full production process, including the route of synthesis for all In-House production as well as the purchased raw materials from qualified vendors was scrutinized and stability of drug substance and drug product of Varenicline tartrate.

Varenicline tartrate is the active pharmaceutical ingredient (API) is used as an aid for smoking cessation¹¹. Varenicline is also used for the treatment of dry eye disease^12-14. The potential for the presence or formation of NDMA, NDEA and N-Nitroso varenicline in the API’s and drug products were reported. So many analytical methods are reported by regulatory agencies to the quantification of nitrosamine impurities in different API’ and dosage forms. To ensure the safety and quality of varenicline tartrate drug substance and drug product (Chantix™), a LC-APCI-MS/MS method has been developed and validated to determine the presence or absence of NDMA, NDEA and N-Nitroso varenicline impurities. The synthetic route of varenicline tartrate and structural information of three nitrosamine impurities (NDMA, NDEA and N-Nitroso varenicline) are shown in (figure 1).

Figure 1: A) Route of synthesis for Varenicline tartrate; B) Nitrosamine Impurities (NDMA, NDEA and N-Nitroso varenicline)

Table 1: Specification calculations of three nitrosamine impurities

Name of Nitrosamine Impurity	Accepted Intake limit (ng/day)	maximum daily dosage (mg/day)	Accepted Intake limit (ppm=AI/MDD)	Taken limits (ppm)
NDMA	96	2	48	13.25
NDEA	26.5	2	13.25	13.25
N-Nitroso varenicline	26.5	2	13.25	13.25

LITERATURE STUDY:

Literature survey revealed, Several LC-HR/MS and LC-MS/MS methods have been reported by the regulatory agencies for determining Genotoxic, Volatile, Organic, nitrosamines in irbesartan, losartan, telmisartan and valsartan drugs etc. and several reports have presented the estimation of nitrosamines in the environment and food by using LC-HRMS and LC-MS/MS^15-36. However, no approach is reported for the trace-level quantification of NDMA, NDEA and N-Nitroso varenicline in the drug substance and drug products by using LC-APCI-MS/MS so far.

The three nitrosamine impurities specifications are calculated by the maximum daily dosage (2mg/day) of varenicline tartrate drug substance and product. The specifications of NDMA, NDEA and N-Nitroso varenicline nitrosamine impurities were calculated by using ‘Accepted Intake limit (AI)’ of three nitrosamines divided by ‘maximum daily dosage (MDD)’ of Varenicline tartrate (2mg/day). The calculated and considered limits are shown in (table 1).

MATERIAL AND METHODS:

Chemicals and reagents:

We have procured all solvents and reagents are LC-MS grade from Honeywell and from Fluka Formic acid was purchased. Varenicline tartrate and two nitrosamine impurities (NDMA, NDEA and N-Nitroso varenicline) were procured from local well-known labs, Hyderabad, India.

Instruments:

Chromatography was performed on Shimadzu LCMS-8045 system, which consisted of a High Performance Liquid Chromatography system coupled to a triple-quadrupole tandem mass spectrometer with an APCI interface. An Inert sustain AQ, C18 150m length × 4.6 mm ID 3.0μm column.

LC-APCI-MS/MS Chromatographic conditions:

LC-MS/MS method was optimized by using gradient elution. 0.1% formic acid in water was used as Mobile Phase A and 0.1% formic acid in methanol was used as mobile phase B. The flow rate was fixed at 0.6mL/min and total run time has 25 minutes. The column oven and auto sampler temperatures were maintained at 40˚C and 15˚C. The injection volume has fixed 30μL. The gradient program (time in min/%B): 0.01/10, 5.00/10, 8.00/40, 13.00/40, 19.00/95, 20.00/10, 25.00/10.

The mass spectrometric conditions were optimized in the APCI positive mode by using the MRM acquisition mode for three nitrosamine impurities. The MRM transitions are shown in (table 2).

Table 2. MRM transitions

Impurity Name	Precursor (m/z)	Product  (m/z)	Q1 Pre Bias (V)	Collision Energy	Q3 Pre Bias (V)
NDMA	75.10	43.20	-6	-11	-44
NDEA	103.20	29.20	-8	-15	-26
N-Nitroso varenicline	241.20	211.10	-12	-13	-14
MS program Valve Position
Initial valve position		1
Time		Command		Valve
8.50		FCV2=		0
13.01		FCV2=		1

APCI source parameters: Nebulizing Gas Flow 3.00 L/min; Interface Temperature 275 °C; DL Temperature: 200ºC; Heat Block temperature 200°C; Drying Gas Flow 3.00L/min respectively. Shimadzu Lab solutions software was used.

Preparation of Solutions:

Diluent:

Prepared the homogeneous mixture of methanol and water in the ratio of 20:80 (v/v) respectively and mix well.

Preparation of Nitrosamine Impurities standard solution (13.25 ppm):

Weighed and transferred about 66.45mg of each nitrosamine impurity in 50mL of volumetric flask and diluted to the volume with diluent and sonicated about 1 min (stock solution-1). Further transferred 5.0mL of stock solution-1 in to 50mL of volumetric flask and diluted with diluent (stock solution-2). Further transferred 0.5mL above stock solution-2 in to 50mL of volumetric flask and diluted with diluent (stock solution-3). Further transferred 0.5mL above stock solution-3 in to 50mL of volumetric flask and diluted with diluent (The 13.25ppm of Nitrosamine Impurities standard solution was prepared with respect to 1.0mg/mL of Varenicline tartrate sample concentration).

Preparation of Varenicline tartrate Drug substance (1.0 mg/mL):

Weighed and transferred 50.15mg of Varenicline tartrate Drug substance into 50mL diluent. Diluted to volume with diluent and mixed the solution using a stir bar and plate until fully dissolved. Filter the solution using a 0.22μm PVDF syringe filter and transfer the filtered sample into an HPLC vial for LC-MS/MS analysis.

Preparation of Varenicline tartrate tablet (1.0 mg/mL):

Crush the appropriate number of tablet(s) to obtain a target concentration of 1.0mg/mL of API in methanol, and transfer into a 15mL glass centrifuge tube. Add the sufficient volume of methanol and mix by using a vortex mixer. Shake the sample by using a mechanical wrist action shaker. Then centrifuge the sample for 15 minutes. Then filter the supernate by using a 0.22μm PVDF syringe and take this solution for LC-MS/MS analysis.

Calculation:

The Nitrosamine Impurities content was calculated from,

ppm=	Impurity area in Varenicline tartrate	×
	Impurity area in Standard solution

Standard Solution Concentration	×	106
Sample Solution Concentration

RESULTS AND DISCUSSION:

Chromatographic method development:

This method development was implemented following Quality-by-Design principles including diluent selection, column selection. During the LC-APCI-MS/MS method development, in order to select the most appropriate system parameters to obtain the best separation, sensitivity and time efficiency, three Nitrosamine Impurities and Varenicline tartrate mixtures were injected under a variety of conditions. Multiple mobile phases with different pH variations and different gradient elutions were used to achieve good peak shapes and better resolution between Varenicline and impurities. After optimization of different trails, the following contentions are given the good peak shapes and sensitivities. Those are, Column is Inert sustain AQ, C18 150 × 4.6mm 3.0μm. 0.1% formic acid in water as Mobile Phase A and 0.1% formic acid in methanol as mobile phase B. The flow rate 0.6 mL/min and total run time has 25 minutes. The column oven and auto sampler temperatures were maintained at 40˚C and 15˚C. The injection volume has fixed 30μL. The gradient program (time in min/% B): 0.01/10, 5.00/10, 8.00/40, 13.00/40, 19.00/95, 20.00/10, 25.00/10.

MS/MS Parameters Optimization:

The main objective of MS conditions optimization is simple, selective and highly sensitive method for the quantification of three Nitrosamine impurities in Varenicline tartrate drug substance and drug product 13.25 ppm impurity solution was used to carry out MS/MS method development. Positive ionization mode was found to be more sensitive than negative ionization mode. The APCI source was used to ionize the impurities. The optimized MS/MS parameters like as, Interface Temperature (°C), Heat Block temperature (°C), DL Temperature (°C), fragmentor voltage (V) and Collision energies for each individual impurity to obtain the better response for product ions.

Method validation:

The LC-MS/MS method has validated as per ICH guidelines³⁷.

Specificity:

A Varenicline tartrate and three impurity standard solutions at specification level prepared and inject into LC-MS/MS system. No interference of Varenicline tartrate drug substance with the three impurities NDMA, NDEA and N-Nitroso varenicline. The RT of three impurities (NDMA, NDEA and N-Nitroso varenicline) 6.55 min, 14.21 min and 17.94 min respectively and Varenicline tartrate sample eluted at 11.80 min. The chromatograms acquired were captured in (figure 2).

Figure 2. A), B) Blank; C) NDMA; D) NDEA; E) N-Nitroso varenicline; F) Varenicline tartrate sample

System precision and Method Precision:

System precision was established by six measurements of the standard nitrosamine impurity solution at the specification level on the same day. Six injections of standard nitrosamine impurity solution were injected into the LC-MS/MS system to evaluate the system precision of developed method. The Relative standard deviation (RSD) was calculated for each nitrosamine impurity. The obtained Mean ± SD of each impurity is not more than 10%.

Method precision was verified by analyzing the Varenicline tartrate drug substance sample was spiked with opted three nitrosamine impurities at specification limit values. Six preparations of standard nitrosamine impurity solution were injected into the LC-MS/MS system to evaluate the method precision of developed method. The relative standard deviation calculated for three nitrosamine impurities was noticed as ≤ 10%, which proved that LC-MS/MS method was precise for evaluation of opted three nitrosamine impurities in Varenicline tartrate drug substance.

The corresponding data (Mean ± SD) for System precision and Method precision is shown in (table 3).

Table 3: System precision and Method precision data for three Nitrosamine Impurities

Parameters	NDMA (Mean ± SD)	NDEA (Mean ± SD)	N-Nitroso varenicline (Mean ± SD)
System precision	177328 ± 4.85	280319 ± 6.85	1167065 ± 1.52
Method precision	178981 ± 2.08	263395 ± 4.55	1200272 ± 3.67

Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD and LOQ concentrations for the three impurities were determined based on S/N ratios of 10.0 for LOQ and 3.3 for LOD respectively. Evaluated the Reproducibility by injecting the six replicate and recovery by triplicate preparations at LOQ level. The data are shown in (table 4).

Table 4: LOD and LOQ data for three Nitrosamine Impurities:

LOQ and LOD Data
Nitrosamine Impurity	LOQ S/N ratio	LOQ Concentration w.r.t sample ( in ppm)	LOQ Avg. Area(n=6)	LOD S/N ratio	LOD Concentration w.r.t sample ( in ppm)		LOQ Avg. Area(n=3)
NDMA	59.56	0.66	8471	32.56	0.22		2827
NDEA	48.78	0.66	14285	36.85	0.22		4535
N-Nitroso varenicline	75.85	0.66	57220	62.53	0.22		18920
Reproducibility and Recovery data at LOQ level
Nitrosamine Impurity	Reproducibility  (Mean ± SD)					Recovery (%)
NDMA	8471 ± 3.98					101.41
NDEA	14285 ± 4.46					93.63
N-Nitroso varenicline	57220 ± 5.28					105.48

Linearity and Range:

Linearity and Range was generated from 0.66 ppm (LOQ) to 19.88 ppm for the three nitrosamine impurities. The calculated Correlation coefficient, slope and intercept values with in the acceptance criteria. Correlation has not less than 0.995 and the data can be seen in (table 5) and the calibration curves are drawn in (figure 3).

Table 5: Linearity data for three Nitrosamine Impurities

Con. (ppm)	NDMA Avg. area (n=2)	NDEA Avg. area (n=2)	N-Nitroso varenicline Avg. area (n=2)
0.66	8826	14912	58080
3.31	45264	74002	291216
6.63	89260	151352	584243
9.94	133504	221701	878009
13.25	178516	293202	1171903
19.88	269339	440738	1767612
Correlation coefficient (r)	1.000	1.000	1.000
SLOPE	13526	21762	88923
Intercept	-201	1627	-3569

Figure 3. Correlation graphs: A) NDMA; B) NDEA; C) N-Nitroso varenicline

Accuracy:

Appropriate amounts of opted three Nitrosamine Impurities were spiked to Varenicline tartrate drug substance sample with preparations (n = 3) at 6.63ppm level, 13.25ppm level and 19.88ppm level of specification quantity value. These spiked samples were analyzed by way of suggested LC-MS/MS method and ascertained the recoveries of three opted Nitrosamine Impurities at every level and obtained recovery range 98.53% - 106.79%. The accuracy of the method was demonstrated by the fact that the recovery for each impurity at each level was within 100±15%.

Robustness:

It assists to find out the effect of slight variations in the different chromatographic conditions. Robustness of the method was checked by varying the flow rate (±0.1 mL/min with 0.6mL/min) and Interface Temperature          (±2°C with 375°C). The standard three Nitrosamine Impurities solutions were injected under each condition into LC-MS/MS. The results were estimated for the mean and standard deviation. The standard deviation is not more than 10% at every changed method parameter for all three impurities.

Ruggedness:

Ruggedness of the LC-MS/MS method was done by the performing the analysis of three Nitrosamine Impurities in six replicates by using different analysts on different days and the results were obtained within the acceptance criteria indicating the method is rugged within the specified range. The %RSD was calculated for day wise and analyst wise as well as individual and cumulative for three Nitrosamine Impurities. The obtained RSD is not more than 10.0% for each impurity.

Solution stability:

Established the solution stability for the Varenicline tartrate sample solution and standard Impurity solution by at 25˚C up to 48hours. Small variation for the three Nitrosamine Impurity solutions. The % variation achieved for three impurities are 100±10%. From the above data, confirmed the stability of impurities in Varenicline tartrate sample solution for at least 48hours.

Pharmacokinetic application:

The validated LC-APCI-MS/MS method was applied to the Drug products of Varenicline tartrate (CHANTIX 0.5mg and CHANTIX 1.0mg). After analysis is completed calculated the three Nitrosamine Impurities content. NDMA and NDEA were not detected and N-Nitroso varenicline is bellowing LOD. These results confirmed the amount of each Nitrosamine Impurity is within the specified limits. No extra peaks which could interfere with the determination of the three Nitrosamine Impurities were observed. Therefore, the proposed method can be confidently employed for the Varenicline tartrate drug products analysis. So, it can be used in the routine quality control of dosage form in industries.

CONCLUSION:

This paper describes a rapid, sensitive and specific LC-MS/MS method developed for the simultaneous quantification of three nitrosamine impurities and complete method validation in Varenicline tartrate drug substance and drug products. This method is very sensitive than the other available methods. Multiple methods have been published for nitrosamines using LC-MS/MS in various drug substances but there is no method was so far reported to quantify theses three nitrosamines in Varenicline tartrate drug substance and drug products using LC-MS/MS till date. The determined LOQ and LOD values are very low with respect to sample concentration which shows the sensitivity performance of the method. The %relative standard deviation of precision was low when compared with ICH acceptance criteria. So, the validated method can be used for routine quantification of all the three nitrosamine impurities in Varenicline tartrate drug substance and drug products.

CONFLICTS OF INTERESTS:

The author declares no conflicts of interest.

ACKNOWLEDGMENTS:

The authors thank management of KVR, KVR and MKR College, Acharya Nagarjuna University, and Guntur for facilities, co-operation and support during the study.

ABBREVAIATIONS:

APCI      - Atmospheric Pressure Chemical Ionization

LC-MS   - Head Space Gas Chromatography Mass Spectroscopy

MRM     - Multi Reaction Mentoring

APIs       - Active Pharmaceutical Ingredients

FDA        - Food and Drug Administration

GTI         - Genotoxic Impurity

ICH        - International Council for Harmonization

LOD       - Limit of Detection

LOQ       - Limit of Quantification

NDMA   - N-nitrosodimethylamine

NDEA    - N-nitrosodiethylamine

RSD        - Relative Standard Deviation

S/N         - Signal to Noise

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Received on 14.04.2023            Modified on 10.08.2023

Accepted on 04.11.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(4):1703-1710.

DOI: 10.52711/0974-360X.2024.00270