Development and Application of Analytical Methods to Quantitate Nitrite in Excipients and Secondary Amines in Metformin API at Trace Levels Using Liquid Chromatography–Tandem Mass Spectrometry

Abstract

Nitrosamine impurities have provoked numerous global medicine recalls due to their possible presence during drug manufacturing or storage. Regarding formulation of nitrosamine impurities, a key risk involves reactions between nitrosating agents (nitrite) in excipients and vulnerable amines as impurities or degradants. Rapid detection across various sample types is essential to support pharmaceutical manufacturing. In this study, two methods were developed to detect nitrite in excipients and crucial secondary amines in active ingredient metformin hydrochloride at trace levels, respectively. The former method was developed based on the reaction of nitrite ions with 2,3-diaminonaphthalene to form 1-[H]-naphthotriazole (NAT), whereas the latter was based on amine tosylation. Mass spectrometric conditions were optimized using electrospray ionization in the positive mode. Multiple reaction monitoring transitions were determined at m/z 170 → 115 for NAT, and m/z 200.1 → 91 for dimethylamine (DMA) and 228.1 → 91 for diethylamine (DEA). These methods were validated using selected eight excipients or metformin hydrochloride in terms of specificity, linearity, accuracy, precision, robustness, limit of quantification (LOQ), and limit of determination according to the ICH guidelines. The results of the validation were within the acceptable criteria. Applicability of the methods was evaluated using 170 pharmaceutical samples donated by industries. The nitrite content in the excipients ranged from <LOQ to 4.74 ppm, with observed levels 1.3 to 6 times higher than the average (0.8 ppm) in the samples. The DMA levels in the metformin hydrochloride were within the limit (500 ppm) but varied significantly (0.2–209.2 ppm) among manufacturers. DEA was detected at lower levels (0.7–0.9 ppm). To mitigate the nitrosamine content in the metformin products, the excipient compositions were investigated by selecting those with low nitrite levels. As the types of impurities detected have become increasingly diverse and detection cycles have become more frequent, the requirement for preemptive safety management to relieve public anxiety is essential for regulatory aspects. Nitrite and secondary amines are crucial precursors to N-nitrosamine, and the suggesting approach could be a means to mitigate N-nitrosamine contamination.

Introduction

In 2018, the European Medicines Agency (EMA) announced the recall of products containing valsartan, an active pharmaceutical ingredient (API) used to treat hypertension, due to the detection of N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) impurities, which are genotoxic carcinogens defined by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) M7 [1,2]. Similarly, other regulatory authorities suspended products containing related sartans from the market and conducted investigations [3,4,5]. Further, NDMA was detected in ranitidine and nizatidine, histamine 2 blockers used for certain gastrointestinal disorders, and metformin (an anti-diabetic agent), in 2019 [6,7,8,9]. Owing to this, global regulatory authorities promptly addressed these findings to protect patients from the risk of ingesting mutagenic carcinogens. South Korea investigated 973 batches of domestically distributed metformin API, as well as 34 imported and 254 locally manufactured drug products. It then banned the sale of 31 products that exceeded its criteria for acceptable intake (AI) [10]. Nitrosamine impurities are potential carcinogens that can be formed during the manufacturing and storage of pharmaceuticals, requiring strict control by regulatory agencies according to ICH M7 [2], even at trace levels, because of their potential risk to humans. The main causes of NDMA or NDEA formation are the reactions of trace amounts of dimethylamine (DMA) or diethylamine (DEA) following the breakdown of solvents (e.g., dimethylformamide) in manufacturing processes, the inclusion of synthetic raw materials, or contamination of the synthetic processes with added or contaminated nitrites under acidic conditions [11]. Recently, a rapid increase in concerns over nitrosamine drug substance-related impurities (NDSRIs) has emerged in global pharmaceutical industries and regulatory authorities [12,13]. NDSRIs are nitrosamines that are structurally similar to the API itself, with the addition of a nitroso (NO) group to form NO-API. These impurities are reportedly formed by reactions between free amines derived from APIs and nitrosating agents (e.g., nitrite); therefore, the control of nitrite or amines in drugs is a significant challenge in the pharmaceutical industry [13]. Korean regulatory authorities have focused on domestically controlling NDSRIs, including N-nitroso sitagliptin amine (NTTP), N-nitrosorasagiline, N-nitrosotamsulosin, and N-nitrosoatenolol.

The U.S. Food and Drug Administration (FDA) and EMA have focused on ensuring the safety of pharmaceuticals by assessing the risks of nitrosamine impurities in APIs and drug products, providing acceptable daily intake limits, and taking necessary measures to reduce or prevent impurities within acceptable levels. The U.S. FDA issued the guidance document “Control of Nitrosamine Impurities in Human Drugs” in September 2020, providing information to mitigate the formation of nitrosamines in drugs [14]. In addition, the agency recently updated this guidance in September 2024, stating that the primary causes of NDSRI formation during manufacturing or the storage of pharmaceuticals are nitrite impurities in excipients Reflecting relevant information, the updated AIs of NDSRIs were provided [15]. Since August 2020, the EMA has provided a general information and Q&A document on nitrosamines to pharmaceutical marketing authorization holders. The document was recently revised in July 2024 to address the principles, methodologies, and scope for managing nitrosamines, including NDSRIs, in pharmaceuticals [16]. To mitigate nitrosamine formation, the aforementioned regulatory bodies recommend monitoring nitrite impurities, qualifying excipient suppliers, and evaluating the potential for nitrite in each batch. Nitrite can also be present in small amounts in various excipients, such as preservatives, dyes, and stabilizers, and can act as a precursor to nitrosamines when it reacts with secondary amines under acidic conditions [16,17,18]. In China, the China National Medical Products Administration (NMPA) also issued technical guidelines for the assessment and control of N-nitrosamine impurities in chemical drugs in 2020. These guidelines outlined potential sources of nitrosamine formation and provided control strategies, including concepts of control, acceptable limits, analytical method development, and risk management throughout the product lifecycle [19]. Furthermore, the 2020 edition of the Chinese Pharmacopoeia incorporated principles for the control of genotoxic impurities, which were consistent with ICH M7(R1) and address the definition, formation pathways, risk assessment, and approaches to setting acceptable limits for genotoxic impurities [20].

Nitrite analysis of pharmaceuticals has commonly been carried out using ion chromatography (IC) equipped with either a conductivity detector or mass spectrometry (MS) [21,22]. The IC system was known to be able to detect and quantify nitrite at low concentrations, in the range of ppm to ppb. In recent studies related to NDMA in drugs, IC-based analysis has frequently been performed to determine levels of nitrite in excipients or water for pharmaceutical use [23,24].

However, according to a domestic survey, there was a limitation that several small companies lack IC instruments to analyze nitrite. As efforts to mitigate NDSRIs, nitrite testing has been required and included in several regulatory guidelines [15,16]. Therefore, pharmaceutical industries such as excipient suppliers and drug manufacturers are recommended to prepare to control nitrite levels in their products. Since MS has become widely adopted in the industries due to nitrosamine testing, most companies maintain liquid chromatography–mass spectrometry (LC-MS/MS) or gas chromatography–mass spectrometry (GC-MS/MS). This MS system can offer the advantage of achieving ppb-level sensitivity for quantitate nitrite, coupling a simple derivatization protocol. The analytical method was selected based on convenience, sensitivity, and feasibility of using internal standards (ISTDs); MS was used for its suitability in isotope measurement, with 15N-nitrite chosen as the ISTD. To enhance analytical systems beyond IC, several studies have focused on the derivatization of nitrite [25,26,27,28,29,30,31,32]. Analytical methods utilizing derivatization reactions to develop quantitative techniques for trace amounts of nitrite present in excipients were reviewed and are summarized in Table 1A. The 1-[H]-naphthotriazole (NAT) method was selected for nitrite analysis because of its stability during derivatization, enhanced accuracy of the analytical results, sensitivity, and selectivity.

Nitrite ions produce the fluorescent substance NAT after reacting with 2,3-diaminonaphthalene (DAN) under acidic conditions. Liquid chromatography (LC) is also considered more suitable than gas chromatography for determining non-volatile nitrites [33]. In the previous study that used LC-MS/MS to analyze nitrite, derivatization was conducted in the presence of sulfuric acid and potassium hydroxide [29]. The drawback of this method is that sulfuric acid is non-volatile and can lead to sulfate formation, potentially causing corrosion or contamination of the ion source or quadrupole of the mass spectrometer. In particular, sulfate can decrease analytical efficiency and cause signal suppression in electrospray ionization (ESI). Therefore, the derivatization method used in this study included hydrochloric acid for reaction initiation and sodium hydroxide for termination; acetonitrile (ACN) was employed to separate the aqueous layer. The selected derivatization process is depicted in Figure 1A.

Metformin hydrochloride is a representative drug substance that contains nitrosamine impurities such as NDMA. It is generally synthesized by reacting DMA hydrochloride and 2-cyanoguanidine, which can produce small residues of DMA in the final product [34]. Secondary amines such as DMA are known to be highly reactive with NO donors to form nitrosamines, especially in acidic environments. This reaction can also occur in basic media under specific conditions, such as photochemical activation or in the presence of catalysts [35]. The residual DMA, acting as a secondary amine, can form NDMA by reacting with nitrosating agents (e.g., nitrite) in the excipients. To evaluate the potential formation of nitrosamines in metformin APIs, an analytical method for the key secondary amines, DMA and DEA, was developed in this study. DEA was additionally selected as the precursor to the potential formation of NDEA owing to its class 1 category as defined in ICH M7 and its regulatory aspects [16]. For DMA, a metformin monograph in the European Pharmacopoeia (EP) was reviewed, in which metformin Impurity F is defined as DEA hydrochloride [36]. The derivatization methods for amines described in the EP include reagents such as dansyl chloride (Dns-Cl), (9H-Fluoren-9-yl) methyl carbonochloridate (Fmoc-Cl), and p-toluenesulfonyl chloride (tosyl chloride; TsCl) (Table 1B) [35,36,37,38,39,40,41,42]. Tosyl chloride was selected for the derivatization reaction because of its high reactivity with the nitrogen atom of amines and its stability in the tosylated amine structure (Figure 1B). To this end, an LC-MS/MS analytical method was developed based on a previous study [38] using tosyl chloride as the derivatization reagent.

Although LC-FLD (fluorescence detector) is commonly used for amine analysis in gas chromatography (GC), IC, and LC, the LC-MS/MS method was selected considering its usability in the industries and to ensure method sensitivity, specificity, and accuracy. Although amines can be analyzed using IC, the use of LC-MS/MS systems, which are already widely implemented in the pharmaceutical industry for NDMA detection, offers practical advantages. By utilizing existing instrumentation, amines can be quantified with high sensitivity without the need for additional analytical equipment.

As previously described, to effectively mitigate nitrosamine formation in pharmaceuticals, concurrent control of nitrite and amine precursors is essential, as they can react under acidic conditions to form nitrosamines. It has been demonstrated that simultaneous quantification of these precursors can improve nitrosamine risk assessment [43].

Analytical methods employing IC have been reported for dual detection of nitrite and amines to mitigate nitrosamines [21,34]. In this context, the aim of this study was to establish highly sensitive and precise analytical methods to identify and quantify nitrite in excipients, which is the primary cause of NDSRIs and nitrosamine formation, as well as to identify the major secondary amines, DMA and DEA, in metformin hydrochloride. As nitrite or secondary amines remain in trace amounts in pharmaceutical excipients or APIs, an accurate and reliable method is crucial for their quantification and identification. Both analytical methods for nitrite or the two amines were developed using LC-MS/MS and validated in compliance with ICH Q2(R1) validation of analytical procedures [44]. The study findings demonstrate the usefulness of mitigating pharmaceutical impurities and provide reliable testing methods for the pharmaceutical industry.

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2. Materials and Methods

Nitrite (99.8%) was purchased from Merck (Darmstadt, Germany), while Nitrite-d₄ (98%, ISTD) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). DMA (100.0%) and DEA (99.59%) were purchased from Chem Service Inc. (West Chester, PA, USA). DAN was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). ACN and water used for nitrite and amine method development were purchased from Merck (Darmstadt, Germany) and Supelco (Bellefonte, PA, USA), respectively. Formic acid (0.1%) and sodium hydroxide were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Boric acid and tosyl chloride were purchased from Sigma Aldrich (Louis, MO, USA). Methanol (99.9%) was purchased from Samchun Chemicals Co., Ltd. (Seoul, Republic of Korea). For method validation, seven excipients [microcrystalline cellulose (MCC), magnesium stearate (MS), colloidal silicon dioxide (CSD), lactose monohydrate (LM), sodium stearyl fumarate (SSF), hypromellose (hydroxypropyl methylcellulose (HPMC)), and sodium carboxymethyl cellulose (CMC)] purchased from Merck (Darmstadt, Germany) were analyzed, in addition to a croscarmellose sodium (CCS) sample donated by a local drug distributor. Eight excipients and metformin hydrochloride APIs donated by domestic drug distributors were used to confirm the applicability of these methods (Table 2). The donated and purchased samples, which were within their shelf life, were used in the experiments. Amber containers were used during all the processes, including sample preparation.

Table 2. List of samples, excipients, and metformin API, donated by 23 pharmaceutical companies.

	Number of Samples (n = 170)	Number of Manufacturers	Number of Manufacturing Countries
Metformin HCl	20	7≤ a	2≤ a
Magnesium stearate	21	3	3
Microcrystalline cellulose	23	5≤ a	5
Colloidal silicon dioxide	18	4≤ a	3
Lactose monohydrate	21	3	2
Sodium stearyl fumarate	14	1	1
Hypromellose (HPMC)	22	5≤ a	4
Croscarmellose sodium	20	6	5
Sodium carboxymethyl cellulose	11	4	4

Ahn, I.; Lee, S.; Jung, M.J.; Jeong, Y.; Kim, J.Y.; Kim, M.; Kim, P.S.; Lee, B.-H.; Lee, Y.M.; Son, K.H. Development and Application of Analytical Methods to Quantitate Nitrite in Excipients and Secondary Amines in Metformin API at Trace Levels Using Liquid Chromatography–Tandem Mass Spectrometry. Chemosensors 2025, 13, 307. https://doi.org/10.3390/chemosensors13080307

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