Simultaneous Detection and Quantification of 12 Residual Solvents in avermectin Sodium Active Pharmaceutical Ingredient by Headspace Gas Chromatography with Flame Ionization Detector

Avibactam is a diazabicyclooctane (DBO) derivative with the chemical name [(1R,2S,5R)–2–(aminocarbonyl)–7–oxo–1,6- diazabicyclo [3.2.1] oct–6–yl] hydrogen sul-fate [1]. Unlike traditional β–lactamase inhibitors, avibactam exhibits a unique, reversible mechanism of action. The inhibition process begins when the serine residue of β–lactamases nucleophilically attacks the amide bond of avibactam, resulting in ring opening and the formation of a covalent enzyme-inhibitor complex [2]. This complex represents the inhibited state of the enzyme, yet unlike classical inhibitors, no hydrolysis occurs. Instead, avibactam can undergo recyclization, reforming its β-lactam ring and re-generating the active molecule. The rate of nucleophilic ring opening significantly exceeds that of recyclization, ensuring that β–lactamases remain predominantly inhibited. Crucially, avibactam’s structure can be restored through reversible reactions, which contributes to its prolonged inhibitory effect [3]. Avibactam demonstrates broadspectrum inhibition against multiple classes of β-lactamases, including class A enzymes (e.g., CTX–M–15, KPC–2), class C enzymes (AmpC), and some class D enzymes (e.g., OXA–48) [4,5]. Clinically, the ceftazidime–avibactam combination has proven effective against Entero-bacteriaceae producing AmpC, KPC, and extendedspectrum β-lactamases (ESBLs) [6].

The synthesis of avibactam and its derivatives involves complex multi-step reactions requiring various organic solvents [7-11]. However, complete removal of organic solvents from final products is often unattainable, and their residual traces may compromise both therapeutic efficacy and patient safety due to inherent toxicity and irritant properties. This necessitates rigorous residual solvent analysis in the quality control of avibactam and its derivatives [12,13]. The International Council for Harmonisation (ICH) Q3C guideline mandates strict control of residual solvents throughout pharmaceutical manufacturing, and the regulation classifies solvents into three categories based on toxicity profiles and establishes permissible exposure limits for each [14]. To ensure compliance and stream-line quality assessment, a robust platform method for residual solvent analysis is crucial. Such a method would serve as a versatile foundation for avibactam production, enabling rapid adaptation to specific program requirements while maintaining analytical con-sistency across different derivatives. However, literature on residual solvent detection in avibactam and its derivative products remains scarce, highlighting a critical gap in cur-rent analytical methodologies for this important class of β– lactamase inhibitors.

(HS–GC–FID) has emerged as the gold standard for residual solvent determination in pharmaceuticals, owing to its exceptional sensitivity, wide dynamic range, and robust performance [15-17]. Ramanjaneyulu et al. established a selective and sensitive HS-GC method for simultaneous analysis of 11 residual solvents (MET, ET, ACE, DCM, MTBE, n-hexane, 1-propanol, EA, THF, DIPEA, and DMF) in rufloxacin hydrochloride. This method demonstrated excellent sensitivity at ppm levels while offering reduced runtime [18]. Similarly, Noorbasha et al. validated a GC method for paclitaxel that simultaneously quantifies MET, ET, ACE, IPA, DCM, n–hexane, EA, THF, and DIPEA. The method proved to be simple, sensitive, and specific, with demonstrated linearity, precision, accuracy, and robustness suitable for both drug substance and product analysis [19]. Gudibanda et al. further advanced the field by developing a novel HS–GC method with enhanced sensitivity for twelve solvents (MET, ET, ACN, IPA, DCM, MTBE, EA, THF, 1,4–dioxane, DMF, hexane fractions, and Tol) in oseltamivir phosphate API and dosage forms [20]. Therefore, based on the synthetic route of avibactam sodium and in accordance with the Chinese Pharmacopoeia (2020 edition) and ICH guidelines, we developed a gas chromatography method for the simple and rapid detection and quantification of over 12 potential residual organic solvents in avibactam sodium and conducted the comprehensive methodological research. This study establishes a technical foundation to ensure the safe production of high–quality avibactam sodium APIs and their derived formulations in future manufacturing processes.

The development and optimization of a generic headspace gas chromatography with flame ionization detection (HS–GC– FID) method for the simultaneous separation of multiple residual solvents is critical for achieving efficient and reliable analytical results during pharmaceutical development [21,22]. In this study, we established a novel gas chromatographic method for the quantitative determination of 12 residual solvents in avibactam sodium active pharmaceutical ingredients (APIs). To demonstrate the method’s applicability for routine analysis of all target solvents, we conducted comprehensive validation in accordance with ICH Q2(R2) guidelines, the Chinese Pharmacopoeia (2020 edition), and current good manufacturing practice (cGMP) requirements [23- 25]. The validation study systematically evaluated key analytical performance characteristics, including specificity, linearity, sensitivity, precision, and accuracy.

Specificity

The specificity test was a critical component of the method development process, designed to verify the optimal performance of the chromatographic system. Blank solvent (1mL), individual reference solutions (1 mL each), and a mixed reference solution (1mL) were prepared and injected into the GC system. The resulting chromatograms were carefully analyzed to determine the retention times of each component and evaluate the degree of separation achieved. This systematic approach ensured the method’s ability to distinguish and quantify the target analytes without interference.

The chromatographic results were presented in Figure 1. The solvents eluted in the following order: methanol (MET), ethanol (ET), acetone (ACE), isopropanol (IPA), acetonitrile (ACN), dichloromethane (DCM), tertbutanol (tBA), methyl tert–butyl ether (MTBE), ethyl acetate (EA), tetrahydrofuran (THF), toluene (Tol), and butyl acetate (BuOAc).

The method validation confirmed excellent specificity, with no interfering peaks detected in either the blank solvent or reference solutions (Figure 1). As summarized in Table 1, all target residual solvents exhibited distinct retention times and achieved baseline separation. Resolution values for all adjacent peak pairs in the mixed reference standard solution exceeded 1.5, demonstrating complete chromatographic separation of the analytes. These validation results confirm that the developed GC method is selective and suitable for the accurate determination of residual solvents in avibactam sodium.

Table 1:The retention time and separation degree of 12 residual solvents.

Table 2:Linear results for each residual solvent (n = 3).

Table 2:Linear results for each residual solvent (n = 3).

Linearity

Each concentration of the linearity solutions was injected in triplicate into the GC system, and the peak areas of the solvents were recorded for each level. The calibration curves were constructed by plotting the concentration (x–axis) against the average peak area (y–axis), followed by linear regression analysis. The solvent peak areas exhibited excellent linearity across the tested concentration range, with correlation coefficients (R2) > 0.999 (Table 2). This strong linear relationship for all 12 residual solvents ensures accurate quantification of residual solvents in avibactam sodium API using the proposed method.

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

The sensitivity of the HS–GC–FID method was determined based on the ICH method, with the LOD defined as a S/N ratio of 3:1, and the LOQ with a S/N ratio of 10:1. As shown in Table 3, the LOD for all residual solvents ranged from 0.30 to 3.66 μg/mL, and the minimum achievable LOD was 0.30 μg/mL for MTBE. The LOQ for all residual solvents ranged from 1.00 to 12.08 μg/mL, and the minimum achievable LOQ was 1.00 μg/mL for MTBE. The results shown that the method established in this study can sensitively detect the residual amount of 12 solvents in in avibactam sodium API.

Table 2:Linear results for each residual solvent (n = 3).

Table 2:Linear results for each residual solvent (n = 3).

Table 2:Linear results for each residual solvent (n = 3).

Table 2:Linear results for each residual solvent (n = 3).

Table 2:Linear results for each residual solvent (n = 3).

Sampling and Laboratory Analysis Techniques

All of the experimental protocols were not need to get ethic approval as there were no experimental animals used.

All of authors consent that this manuscript was published in this journal.

No consent to participate.

No Conflict of interest.

All data generated or analyzed during this study are included in this published article.

Conflict of Interest: The authors declare no competing interest.

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