Abstract

Lercanidipine is a synthetic antihypertensive drug belongs to the dihydropyridines chemical class. Antihypertensive drugs often require long term usage for controlling blood pressure and to prevent cardiovascular risk. Drugs that possess secondary amine functional group are prone to nitrosation and form nitrosamine drug substance related impurities (NDSRI). Nitrosamine impurities are known to cause carcinogenicity in humans and required to be controlled in pharmaceuticals. The present work describes rapid 5.0 minute liquid chromatography mass spectrometry method for the detection and quantitation of potential genotoxic Nitrosamine impurity N-nitroso-des-methyl-lercanidipine impurity D (NNLD) in the Lercanidipine active pharmaceutical ingredient. The developed method validated as per ICH Q2(R2) guideline and shown excellent selectivity, linearity (r² 0.9957), accuracy (average % recovery 90 to 111 %) and precision (%RSD < 8%). The Limit of Detection (LOD) was 0.17 ppm and Limit of quantification was established at 0.5 ppm relative to 5mg per mL Lercanidipine test concentration.

1. Introduction

Lercanidipine(LD) chemically called 5-O-[1-[3,3-diphenylpropyl(methyl)amino]-2-methylpropan-2-yl] 3-O-methyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate is a synthetic antihypertensive drug belongs to the dihydropyridines chemical class ^{1 2 3}. Hypertension is chronic disease and requires long term treatment with anti-hypertensive drugs ⁴. Drugs and their impurities that possess secondary amine functional group are prone to nitrosation and form Nitrosamine drug substance related impurities (NDSRI) ^{5 6 7 8 9}. Nitrosamine impurities are known to cause carcinogenicity in humans and are required to be controlled and monitored at trace levels in pharmaceuticals ^{10 11}. Lercanidipine related impurity referred as Dehydro lercanidipine is prone to nitrosation and form Nitrosamine related impurity called N-nitroso-des methyl-lercanidipine impurity D (NNLD). The acceptable intake value proposed by European medicines agency (EMEA) as per appendix-1 EMA/42261/2025/Rev. 13 updated on 24-JUN-2026 is 100ng/day for NNLD ¹² calculated using Carcinogenic potency categorization approach ¹³. The maximum recommended daily dose (MDD) for LD is 20mg ². Based on MDD the specification limit for NNLD is 5 µg/g (ppm).

The objective of the current study is to develop and validate reliable Liquid chromatography coupled with triple quadrupole mass spectrometric (LC-MS/MS) analytical method for the trace level detection and quantitation of N-nitroso-desmethyl-lercanidipine impurity D (NNLD) in Lercanidipine drug substance.

Literature survey revealed few analytical methods for Lercanidipine and its metabolites in biological fluids using LC-MS/MS technology ^{14 15 16 17}. Few more methods are reported for Lercanidipine and its related impurities in active pharma ingredients and dosage forms using liquid chromatography coupled with UV detection techniques ^{18 19 20}. To the best of our knowledge no methods were available for the detection and quantitation of NDSRI impurities in Lercanidipine. The current paper presents novel LC-MS/MS (ESI+Ve MRM mode) validated method with shorter run time of 5 minutes for the detection and quantitation of NNLD in active pharmaceutical ingredient at a detection limit of 0.17 ppm relative to 5 mg/mL LD in test solution.

2. Materials

LC-MS grade solvents methanol, acetonitrile, HPLC grade water purchased from Honeywell local suppliers. Ammonium formate and formic acid were purchased from Merck local suppliers. Lercanidipine (LD) Active pharmaceutical ingredient (API) N-nitroso-desmethyl-lercanidipine impurity D (NNLD) (Rotamers) were provided as gift samples from Svak life sciences, Hyderabad India.

Ultra high performance liquid chromatographic system combined with triple quadrupole Quattro Premier XE mass spectrometer from Waters Corporation, Milford, MA 01757, was used for experiments. Data acquired and processed using Mass Lynx version 4.1 software. Data processed using Spectrus® 2025 2.1 software from ACD/Labs (Company of Revvity Inc, USA).

3. Methods

Separation of impurity NNLD from the Lercanidipine achieved on HyPURITY C18(50mm length, 4.6mm id and 5µM particle size) from Thermo Fisher scientific. The retention times were 2.70 minutes for LD and 3.95 minutes for NNLD. Mass spectrometer operated in electro spray positive ionization mode (ESI+ve). Analytes were subjected to mass fragmentation and Multiple reaction monitoring mode (MRM) used for signal acquisition and quantitation. Detailed LC-MS conditions were described in Table 1.

0.5 mg/mL Impurity stock solution prepared by dissolving impurity NNLD in methanol with the aid of sonication. Further dilutions were made using 50:50%v/v water: methanol as diluent to prepare working standard solutions in the concentration range of 10 to 130% of the specification limit i.e. 5 ppm relative to 5 mg/mL Lercanidipine in test solutions. The absolute concentration of NNLD range from 2.5 ng/mL to 32.6 ng/mL.

5 mg/mL Lercanidipine test solution prepared by dissolving and diluting LD active pharmaceutical ingredient (hydrochloride salt) with 50:50%v/v water: methanol as diluent using sonication. Accuracy and precision solutions were prepared by spiking appropriate volumes of NNLD impurity working standard solutions.

4. Results and Discussion

Lercanidipine and its NDSRI impurity NNLD are hydrophobic in nature, hence method development trials started on C18 column chemistry with different pH buffers (0.1% formic acid, 5mM Ammonium formate and formic acid, 5 mM Ammonium acetate) and using acetonitrile as organic modifier. Good chromatographic resolution greater than 3.0 achieved with 5mM Ammonium formate buffer with 0.2% formic acid buffer. This separation allows to divert high concentration of Lercanidipine API into waste to prevent contamination of MS detector. This buffer also shown good ionization for NNLD impurity and thereby giving good sensitivity. Gradient, column temperature, flow rate parameters were further optimized to reduce runtime and better peak shape. Mass spectrometric parameters were optimized through tuning experiments by injecting NNLD impurity solution and systematically varying instrumental settings. NNLD Precursor ion (m/z 625) subjected to fragmentation with different collision energies and observed stable and intense fragments observed at 30.0 eV collision energy. Most intense fragments; m/z 295 and m/z167 were selected as Quantifier and qualifier ions respectively.

Note: NNLD standard used is a mixture of two rotamers and observed shoulder peak due to rotamer and considered for quantification

The developed method validated in accordance with ICHQ2(R2) guideline ²¹. Specificity, linearity, accuracy, Precision, Intermediate precision, LOQ, LOD, range parameters were evaluated and all the validation results are well within the acceptable limits.

Specificity for the method was established by injecting and comparing diluent, LD drug substance matrix solution and LD drug substance solution spiked with NNLD impurity. No interference was observed at selected MRM transitions (m/z 625 to m/z295) in the blank and drug matrix at the retention time of NNLD. In addition, Quantifier(m/z295) to qualifier (m/z167) are well with in ± 30% deviation from the ratios established for standards covering the range of 10% to 120% of specification limit (0.5ppm to 6.0 ppm relative 5 mg/mL LD).

The correlation between quantifier ion response (m/z 295) and concentration was evaluated using a series of NNLD impurity solutions prepared from the stock solution. Linearity was assessed across seven concentration levels, ranging from 0.5 ppm (10% of LOQ) to 6.0 ppm (120% of the specification limit), relative to 5 mg/mL LD in the test solution. The calibration curve demonstrated excellent linearity over this range, with results summarized in Table 2.

Method reliability was checked by performing recovery studies. NNLD impurity stock solution was spiked at different concentration levels (10%, 50%, 100%, 120% of the specification limit) into the 5 mg/mL LD test solution and %recovery for NNLD calculated. Accuracy and Method precision was studied using 6 replicate preparations of spiked solution at 10% and 100% levels of the specification limit. Accuracy at 50% and 120% levels was studied using 3 replicate preparations.

Intermediate precision was checked at 100% level using 6 replicate preparations done by different analyst on different day. The overall %RSD (n=12) less than 10% indicates method repeatability on different days with different analysts.

The average %recovery values between 90% to 111% across all the levels indicates method was accurate enough to quantify NNLD impurity from drug substance test solutions from 0.5 ppm to 6.0 ppm relative to 5 mg/mL Lercanidipine.

The %relative standard deviation (%RSD) for the NNLD content in 6 replicate preparations at 10% and 100% levels was less than 8% indicated good method repeatability. Accuracy and precision results were reported in Table 3.

The developed analytical method was linear and accurate in the range of 2.5 ng/mL to 30 ng/mL of NNLD impurity absolute concentration and 0.5 ppm to 6 ppm relative to 5 mg/mL LD in drug substance solutions.

The limit of quantification (LOQ) for the developed method was found 0.5 ppm relative to 5 mg/mL LD in test solution with signal to noise ratio greater than 10 for NNLD peak with acceptable accuracy and precision. The limit of detection (LOD) with signal to noise ratio greater than 3 was established at 0.17 ppm. At 0.17 ppm concentration level NNLD signal can be detected and differentiated from baseline noise.

Solution stability was evaluated for 24 hours at 10°C by keeping solutions in amber glass vials. The relative %change in NNLD content is less than 12% compared to initial results. This indicates solutions were stable for 24 hours at 10°C with light protection.

5. Conclusion

The method validation results indicate that the developed method can be successfully applied for the detection and quantitation of Nitrosamine related impurity: N-nitroso-des methyl-lercanidipine impurity D (NNLD) in Lercanidipine drug substance batches. The developed method with shorter run time (5.0 minutes) reduces resource consumption and enhances productivity during routine quality control testing of lercanidipine drug substance.

ACKNOWLEDGEMENTS

The Authors are thankful to the Andhra University College of Pharmaceutical Sciences, Visakhapatnam, Andhra Pradesh, India, for their support in conducting this experimental work. We also acknowledge Svak Life Sciences, Hyderabad, Telangana, India, for providing the drug substance and NDSRI impurity as gift samples.

References