Development of GC-MS/MS Method for Simultaneous Estimation of Four Nitrosoamine Genotoxic Impurities in Valsartan

Objectives: Recently, N -nitrosamines were unexpectedly detected in valsartan and other generic sartan products. Taking into this account, we developed a sensitive and stable multiple reaction monitoring mode-based “gas chromatography-tandem mass spectrometry (GC-MS/MS)” approach for the quantification of “four N -nitrosamines” in valsartan, especially, N -nitrosodiisopropylamine, N -nitroso ethyl isopropylamine, N -nitrosodiethylamine, and N -nitrosodimethylamine. Materials and Methods: GC and MS conditions were optimized with specificity, sensitivity, linearity, precision, and accuracy of the parameters. The approach was validated as per the “International Council for Harmonization” recommendations. Results: The identification limits and limits of quantification of N -nitrosamines in valsartan varied between 0.02 and 0.03 ppm, and 0.06-0.09 ppm, respectively. The obtained values were satisfactory with limits established by the United States Food and Drug Administration for sensitivity requirements. The regression coefficients greater than 0.999 for four N -nitrosamines in the calibration curve demonstrated the strong linearity of the process. The retrievals of “ N -nitrosamines” in valsartan between 91.9-122.7%. For the intra-day and inter-day accuracy studies, the (relative standard deviation) was less than 9.15%. Conclusion: The proposed approach has rapid analysis capability, high precision, accuracy, and good sensitivity, which give a reliable approach for N -nitrosamines quality control in valsartan.


INTRODUCTION
In the production process of the "active pharmaceutical ingredients (APIs)", impurities are incorporated through various sources, like catalysts, solvents, reagents, intermediates, starting materials, and by-products. Compared to other impurities, genotoxic impurities (GTIs) are of a special kind that could inspire mutations at the genetic level, which lead to chromosomal breakage and rearrangements, and even present in low concentrations have increased the risk of cancer. 1 Considering into account the toxic effects of GTIs the international regulatory bodies had set an exposure limit thresholds for GTIs, specifically, 1.5 μg/day for long-run therapy with greater thresholds of clinical shorter intervals. As these GTIs are present in very low concentrations, the pharmaceutical industry faces an uphill task of developing robust analytical, sensitive, and high efficient methods for their determination. 2,3 Valsartan belongs to the category of antihypertensive drugs that selectively inhibit angiotensin receptor type II. It is used to treat mild-to-moderate essential hypertension. The angiotensin-II mediated unwanted effects are reduced to a significant extent by valsartan. Recalls for valsartan were issued between mid-to-late 2018. The cause of the recalls was due to the detection of GTIs such as "N-nitrosodiethylamine (NDEA)" or "N-nitrosodimethylamine (NDMA)" in valsartan in unacceptable limits. 4 The nitrosamine impurities were produced during the drug substance synthesis in which the sodium azide, which is applied in the production of the tetrazole moiety, was eliminated using sodium nitrite and later under acidic circumstances would form nitrous acid, a powerful nitrosylating agent. Dimethylamine and diethylamine, which might be present as impurities in dimethylformamide may be N-nitrosylated in the synthesis might result in the formation of NDMA and NDEA. Likewise, in certain production processes for valsartan, the reagent triethylamine can degrade to produce diethylamine, and latter N-nitrosylated to produce NDEA. N-nitroso compounds are included in the "cohort of concern" as they exhibit great carcinogenic potency. 5,6 The appropriate regular intake limits for NDEA, NDMA, and other impurities in some products were published by the United States Food and Drug Administration (FDA). The limit of N-nitrosoethylisopropylamine (NEIA) and N-nitrosodiisopropylamine (NDIPA) is 96 ng/day.
Recently, for detecting N-nitrosamines in water, food, and personal care products, the analytical techniques widely used are gas chromatography (GC), 7-18 liquid chromatography (LC) 19,20 and supercritical fluid chromatography (SFC)-tandem mass spectrometry (MS). 21 FDA has determined the interim appropriate regular intake levels for N-nitrosamines in valsartan (Table 1) and employed GC-MS/MS by using liquid injection and headspace, 22,23 rapid fire-MS/MS, 24 and high performance liquid chromatography (HPLC)-high resolution mass spectrometry 25 for quantification of the N-nitrosamines in valsartan.
In the analysis of water, food, and personal care items, the extraction as well as purification measures are critical and important. However, in the pharmaceutical industry, these were much not much applicable. Liu et al. 26 reported a GC-MS/MS approach for detecting four "N-nitrosamines" in valsartan, but the method suffers from the drawback of long run time and less accuracy. Therefore, we established through direct injection for quantification of four "N-nitrosamines", as a simple, sensitive, precise, and rapid GC-MS/MS approach for valsartan. The limit of detections (LODs) and limit of quantification (LOQ) values were at acceptable limits as per the sensitivity requirements set by the FDA. The proposed GC-MS/MS approach validation was performed as per the International Council for Harmonization (ICH) guidelines.

Instrumentation and optimized GC-MS/MS conditions
Agilent "7890B" "GC-MS/MS" equipped with an "Agilent 7693A" auto sampling device and 7697 a Headspace Sampler was used to examine N-nitrosamines. The analytical column used was DM-WAX (30 m x 0.25 mm, 0.5 μm). This detection was conducted at a 700°C triple quadrupole mass spectrometer consisting of electron ionization (EI) ion source. The temperature programming in GC oven was done by maintaining oven temperature of 70°C for 4 min, which first elevated at 20°C·min -1 to 240°C and maintained for 3 min. The run interval was fixed at 10 min. The flow rate was 3.0 mL/min for helium as the carrier gas. The injection temperature as well as injection interface were maintained at 240°C. The volume of injection in the split mode (1:2) was 1 μL. MS was run at 70 eV in EI mode with a 150°C "quadrupole temperature". The ion source has been adjusted to a temperature of 230°C. It was 4 min to delay the solvent. The data recovery mode for the quantitative estimation of "four N-nitrosamine GTIs" was chosen as the "multi-reaction monitoring (MRM)" mode. Table 2 provides a summary of the data for precursor ions, production and enhanced "collision energy" for four N-nitrosamine GTIs.

Preparation of samples and standard solutions
The standard stock solutions of NDMA, NDEA, NEIA, and NDIPA were prepared at 1 mg/mL concentrations through dissolving weighed reference standards in 1-methyl-2-pyrrolidinone, respectively, and stored at 4°C. A sequence of standard working

Method validation
The developed "GC-MS/MS" approach with MRM mode for detecting four N-nitrosamines was validated for parameters, as solution stability, precision, accuracy, LOQ, LOD, linearity, sensitivity, specificity, and system suitability. For LODs, signalto-noise (S/N) ratio was 3 as well as LOQs were S/N: 10. To the accuracy of the proposed method, the recovery studies were conducted for evaluation. The precision studies were evaluated by inter-day and intra-day relative standard deviations (RSDs) of six specimens spiked across 3 continuous days at a single concentration. There was no statistical analysis performed in the developed method.

Selection of solvents
Considering the trace level of N-nitrosamine GTIs NDMA, NDEA, NEIA, and NDIPA in valsartan and solubility parameter, 1-methyl-2-pyrrolidinone was selected as a solvent for the preparation of solutions for GC-MS/MS analysis.

Capillary column selection
Under the given temperature program, three different capillary columns were used to obtain the best chromatographic separation. The columns were HP-35MS, HP-5MS, and DM-WAX in the order of increasing polarity. A 10 μg/mL normal blend was inserted in all three cases. Identification of the compounds depended on the spectra EI using the National Institute of Standards and Technology library. Liu et al. 26 reported separation of four nitrosamines using DB-WAXetr capillary column without the inclusion of NDEA. We have used the DM-WAX column, which has sufficient polarity to isolate all nitrosamines with better peak shapes, resolution, less analysis time with applicable for the analysis of most polar and volatile compounds, such as NDMA and NDEA.The optimized chromatogram is shown in Figure 2.

Mass spectrometry
In the analysis of pharmaceuticals, the most crucial aspect is the trace detection method for GTIs. Considering the sensitivity criteria, MRM mode is superior to SIM mode for the quantification of N-nitrosamines. Consequently, the MRM mode was used to quantify four N-nitrosamine GTI in valsartan as MS approach. MS of valsartan and four N-nitrosamine GTIs with chromatograms are shown in Figure 3.

Method validation
The suggested approach for "four N-nitrosamine GTIs" was validated as per ICH recommendations.

Specificity
For the specificity of the recommended approach, 1-methyl-2-pyrrolidinone, the valsartan matrices and mixed standards of four N-nitrosamines were undergone by the "GC-MS/MS" examination. At the retention times of four N-nitrosamines there is no interference peaks from the 1-methyl-2-pyrrolidinone, and the valsartan matrices were observed, indicating the specificity of the approach for detecting four N-nitrosamines in valsartan.

Linearity and sensitivity
The data for linearity, LOQ, and LOD outcomes are summarized in Table 3. The standard curve (y= Ax + B, in which A signifies the slope and B signifies the intercept) was obtained by plotting the chromatographic peak area (N-nitrosamines, y) to normal N-nitrosamines (x) concentrations. To validate the linearity of the approach, six standard concentrations were used. The regression coefficients (R 2 ) of the standard curve for four N-nitrosamines were >0.99 in the given concentration range, indicating better linearity and is appropriate for the quantitative examination, as shown in Figure 4. Therefore, based on LODs and LOQs, the sensitivity of the method was evaluated. In Table 3 Table 4. Considering the ultra-trace essence of the study, the recovery of N-nitrosamines was found in the acceptable range of 70-130%, indicating the accuracy of the proposed method for N-nitrosamines.

Stabilities of four N-nitrosamines in 1-methyl-2-pyrrolidinone
To study four N-nitrosamines solution stabilities in 1-methyl-2-pyrrolidinone, 0.3 ppm standard solutions were prepared and analyzed every 4 h for a recently prepared standard. Each solution was placed at 25°C in the dark. The recovery percentage of N-nitrosamines from these stock solutions was between 97.51 and 105.04%, and the differential recoveries of N-nitrosamines at 0 h and 24 h at just 10%, indicate the stability of the stock solution for at least 24 h.

Applications in samples
This GC-MS/MS process was used in the determination of four N-nitrosamine GTIs in four batches of commercial valsartancontaining products and none of the four "N-nitrosamines" were observed in four batches of the commercially available formulation.

Ethics
Ethics Committee Approval: Not required.
Informed Consent: Not applicable as our study does not involve any human volunteers and animals.