Development of an LC–MS Method for 4-Fluoroaniline Determination in Ezetimibe

Development of an LC–MS Method for 4-Fluoroaniline Determination in Ezetimibe Abstract A rapid and sensitive high-performance liquid chromatography–mass spectrometry method was developed and validated to determine 4-fluoroaniline concentration in ezetimibe. Chromatographic separation was achieved on a Phenomenex Gemini-NX C18 column (150 × 4.6 mm, 3 μm) maintained at 30°C. The liquid chromatography system was operated in gradient mode with an injection volume of 20 μL at a flow rate of 1 mL/min. Mobile phase A was water and mobile phase B consisted of acetonitrile with 0.05% acetic acid. The detection was performed using a single quadrupole mass spectrometer in single ion monitoring mode by using positive ionization. An m/z value of 112 was selected for monitoring 4-fluoroaniline. The method showed good linearity over the concentration range of 0.94–30.26 ng/mL. The limit of quantification and limit of detection were 0.19 and 0.94 ng/mL, respectively. The precision relative standard deviations were less than 8.7% (n = 12), and the accuracy values were within 92–99%. A standard solution of 4-fluoroaniline was stable for at least 24 h at 25°C. Small changes in the organic phase acidity of the mobile phase, flow rate, column temperature, and the instrument parameters had no significant effect on the results for 4-fluoroaniline. Introduction The widespread use of aniline and its derivatives in the pesticide, plastic, and pharmaceutical industries has led to the contamination of aquatic environments and drug products with its chemical by-products. These compounds are well known due to their high toxicity and suspected carcinogenicity (1–4). Aniline compounds can be ingested, and this causes a decline in the oxygen-carrying capacity of hemoglobin, resulting in animal poisoning. In humans, aniline inhalation or absorption results in acute poisoning or chronic intoxication characterized by symptoms such as headaches, dizziness, drowsiness, memory loss, breathing difficulties and asphyxia. Long-term exposure to aniline compounds may induce cancers. The presence of residues of aniline in the environment has garnered attention that has resulted in its long-term inclusion on a list of contaminants requiring priority monitoring (5). Since anilines contain a benzene ring with a π conjugated system that can absorb ultraviolet (UV) radiation, UV determination and high-performance liquid chromatography (HPLC) can be employed for detection. Common techniques for the determination of aniline and its derivatives are spectrophotometry and chromatography. Initially, a diazo-coupling colorimetric method, using N-(1-naphthyl)ethylenediamine, was primarily used for the detection of aniline compounds through spectrophotometry at 545 nm. However, this method has relatively rigid requirements regarding the temperature of the chromogenic reaction and can produce unwanted reactions. However, most aniline compounds have low boiling points, and thus gas chromatography (GC) can be used to perform qualitative and quantitative studies. Although the GC method is easy to perform and has high specificity, it is not applicable to the study of compounds that produce aniline substances by high-temperature degradation. The constant evolution of science and technology has resulted in the emergence of more efficient and sensitive detection techniques that can be used to study residual amounts of aniline compounds. Some of these techniques are GC–tandem mass spectrometry (GC–MS-MS), liquid chromatography–tandem mass spectrometry (LC–MS-MS), liquid chromatography–nuclear magnetic resonance spectroscopy (LC–NMR), supercritical fluid chromatography (SFC), MS, electrochemical methods and nuclear magnetic resonance spectroscopy (NMR) (6–10). In 2002, Bundy et al. studied the metabolism of 4-fluoroaniline by Eisenia veneta and performed a quantitative study of its metabolic products by using HPLC coupled with 19F/1H–NMR (11, 12). Shuhui et al. established a capillary zone electrophoresis (CZE) with field-enhanced sample injection method for the direct determination of aniline and its derivatives in environmental water. The method was simple, sensitive, and environmentally friendly (8). In the aforementioned methods, little reference was made to the quantification of trace residual 4-fluoroaniline or determination of 4-fluoroaniline in thermally unstable drugs. 4-Fluoroaniline is a common fluorine-containing aromatic amine, widely used in the synthesis of medicines and pesticides, such as the drug ezetimibe (used to treat primary hypercholesterolemia), the drug norfloxacin (a broad-spectrum antibacterial agent), and a 4-amidogen quinoline pesticide. Ezetimibe is a selective inhibitor of intestinal cholesterol and phytosterol absorption, and is often used in combination with simvastatin, an HMG-CoA reductase inhibitor. Ezetimibe, the first specific inhibitor of the intestinal cholesterol uptake transporter Niemann-Pick C1-Like 1 (NPC1L1) protein, was developed as an agent to lower plasma levels of low-density lipoprotein cholesterol (LDL–C) (13, 14). 4-Fluoroaniline is used as a starting material in the synthesis of ezetimibe and can remain in the final product as an impurity (Figure 1). The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) indicates that all compounds with aniline structures have potential genotoxicity, which warrants rigid control of their residual content in drugs. A threshold of toxicological concern (TTC) value of 1.5 μg of a mutagenic impurity per person per day is considered a negligible risk (theoretical excess cancer risk of <1 in 100,000 over a lifetime of exposure) and can be used for most pharmaceuticals as a default to derive an acceptable limit (15–20). From this calculation, considering the daily dose of ezetimibe to be ≥10 mg, the acceptable residual limit of 4-fluoroaniline is 0.015% (w/w). Thus far, there have been no studies on the determination of 4-fluoroaniline in drugs. This article reveals the findings of the first, thorough investigation of the small amounts of this genotoxic impurity, which is present in ezetimibe as a residual from the synthesis process, using a highly sensitive LC–MS method for detection. Figure 1. View largeDownload slide The synthesis and degradation processes of ezetimibe. Figure 1. View largeDownload slide The synthesis and degradation processes of ezetimibe. Materials and Methods Chemicals and materials Acetonitrile of LC–MS grade was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol of LC–MS grade was purchased from Merck (Darmstadt, Germany). Formic acid of analytical grade (97%) was obtained from Alfa Aesar (Tianjin, China). HPLC-grade trifluoroacetate was purchased from J&K Chemical Ltd. (Shanghai, China). Ammonium formate (≥99.999%) was purchased from Sigma-Aldrich. Ammonium acetate (≥98%) and glacial acetic acid (99.5%) were purchased from Sigma-Aldrich. Milli-Q water was of ultra-pure quality (>18 MΩ/cm) and prepared in-house. Nitrogen (99.999%) was purchased from Shengtang (Tianjin, China). 3-Fluoroaniline, 4-fluoroaniline and aniline were all of >95% purity and were purchased from Alfa Aesar (Tianjin, China). Ezetimibe was provided as a gift sample by DEYUAN Pharmaceutical Ltd. (Lianyungang, China). Instrumentation GC–MS system The GC–MS system consisted of a Shimadzu GCMS-QP2010 Plus gas chromatography–mass spectrometer and Agilent 7694E headspace gas chromatography injection system. The system was operated using the Shimadzu GC–MS solution software operation system. The chromatography was performed using a 0.25 μm, 30 m × 0.25 mm (i.d.) capillary column (DB-5MS). The carrier gas was nitrogen, and the flow rate was 1 mL/min (constant). The oven temperature was initially 60°C and was kept at this level for 5 min. It was then increased to 140°C at the rate of 10°C/min, kept constant for 1 min, subsequently increased to 220°C at the rate of 30°C/min, and finally kept steady for 1 min. The inlet temperature was 250°C. The ion source temperature was 230°C. The mass spectrometer used for analysis was equipped with an EI source and data acquisition was carried out in single ion monitoring (SIM) mode. The following conditions were used in the headspace instrument: the injection volume was 1 mL using headspace injection mode with a split made in a 10:1 ratio, the headspace equilibrium temperature was 110°C, loop temperature was 120°C, and transfer line temperature was 130°C. The total run time of the method was 7.4 min. LC–MS system The LC–MS system consisted of an Agilent 1260 with a G1379B 1260u-Degasser, G1312B 1260 Bin Pump system, G1316C 1260 TCC column oven and a G4212B 1260 DAD detector connected to an Agilent 6130 MSD mass spectrometer. The system was operated using Agilent ChemStation software. The chromatography was performed using a 3 μm 110 A, 150 × 4.6 mm (i.d.) C18 column (Phenomenex Gemini-NX). The liquid chromatography system was operated in a gradient mode with a flow rate of 1 mL/min. Solvent A was water and solvent B consisted of acetonitrile with 0.05% acetic acid. The linear gradient started at 10% solvent B and increased to 80% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. Starting conditions were achieved in 1 min and the column was re-equilibrated for 5 min. The injection volume was 20 μL and the column oven temperature was 30°C. Needle wash was performed before and after injection. The total run time was 15 min. In the mass spectrometer, positive electrospray ionization (ESI) was used. Data acquisition was carried out in SIM mode using positive ionization; nitrogen was used as sheath, auxiliary and ion sweep gas. Applied spray pressure was 35 psi, drying gas flow was 10 mL/min, drying gas temperature was 350°C, and fragment was 80 V. Preparation of standards and samples GC–MS Standard stock solutions of 4-fluoroaniline were prepared in methanol at a final concentration of 1.5 mg/mL. The 4-fluoroaniline stock solution was diluted in methanol to achieve a final concentration of 15 ng/mL. Sample solutions of ezetimibe were prepared in methanol at final concentrations of 0.1 mg/mL, and were injected into the GC–MS system after filtration through 0.45 μm nylon filters. LC–MS Standard stock solutions of 4-fluoroaniline were prepared in acetonitrile at a final concentration of 1.5 mg/mL. These solutions were stored at 2–8°C until use. The 4-fluoroaniline stock solution was diluted in acetonitrile to achieve a final concentration of 15 ng/mL. Standards of 4-fluoroaniline, to be used for the preparation of calibration curves, were prepared in acetonitrile by serial dilution over the concentration range 0.94–30 ng/mL. Mixed standard solutions of 4-fluoroaniline, o-fluoroaniline, 3-fluoroaniline and aniline were prepared in acetonitrile at a final concentration of 15 ng/mL. Sample solutions of ezetimibe were prepared in acetonitrile at a final concentration of 0.1 mg/mL and injected into the LC–MS system after filtration through 0.45 μm nylon filters. Development and optimization of chromatographic analysis GC–MS Possible residual 4-fluoroaniline in ezetimibe was detected by applying the GC–MS method according to published protocols, as well as information regarding the physical and chemical properties of 4-fluoroaniline (21). The detailed GC–MS conditions were given in GC–MS system. LC–MS Optimization of the column One milliliter of mixed standard solution was transferred to a flask, and used to test individual chromatographic columns with different specifications. Examples included Phenomenex Gemini-NX C18 110 A (150 × 4.6 mm2, 3 μm), Phenomenex Kinetex C18 (150 × 4.6 mm2, 2.5 μm), Waters Sunfire C18, (150 × 4.6 mm2, 3.5 μm), and Waters XBridge C18, (150 × 4.6 mm2, 5 μm). The liquid chromatography system was operated in a gradient mode with a flow rate of 1 mL/min. Solvent A was water and solvent B consisted of acetonitrile with 0.05% acetic acid. The linear gradient started at 0% solvent B and increased to 60% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. Optimization of the mobile phase Six different mobile phases (cases a–f, Table I) were selected and tested to optimize the composition of the mobile phase. Chromatographic separation was achieved on a Phenomenex Gemini-NX C18 column (150 × 4.6 mm2, 3 μm) maintained at 30°C, following a gradient program. In each case, the linear gradient started at 0% solvent B and increased to 60% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. Table I. Mobile Phase Composition Used in the LC–MS Method Development Case  Mobile phase composition  A  B  a  10 mM ammonium formate with pH adjusted to 4.5  Acetonitrile  b  10 mM ammonium acetate with pH adjusted to 6.7  Acetonitrile  c  0.004% formate with pH adjusted to 3.4  Acetonitrile  d  0.02% formate with pH adjusted to 3.0  Acetonitrile  e  0.1% acetate acid with pH adjusted to 3.2  Acetonitrile  f  H2O  Acetonitrile with 0.05% acetic acid  Case  Mobile phase composition  A  B  a  10 mM ammonium formate with pH adjusted to 4.5  Acetonitrile  b  10 mM ammonium acetate with pH adjusted to 6.7  Acetonitrile  c  0.004% formate with pH adjusted to 3.4  Acetonitrile  d  0.02% formate with pH adjusted to 3.0  Acetonitrile  e  0.1% acetate acid with pH adjusted to 3.2  Acetonitrile  f  H2O  Acetonitrile with 0.05% acetic acid  Table I. Mobile Phase Composition Used in the LC–MS Method Development Case  Mobile phase composition  A  B  a  10 mM ammonium formate with pH adjusted to 4.5  Acetonitrile  b  10 mM ammonium acetate with pH adjusted to 6.7  Acetonitrile  c  0.004% formate with pH adjusted to 3.4  Acetonitrile  d  0.02% formate with pH adjusted to 3.0  Acetonitrile  e  0.1% acetate acid with pH adjusted to 3.2  Acetonitrile  f  H2O  Acetonitrile with 0.05% acetic acid  Case  Mobile phase composition  A  B  a  10 mM ammonium formate with pH adjusted to 4.5  Acetonitrile  b  10 mM ammonium acetate with pH adjusted to 6.7  Acetonitrile  c  0.004% formate with pH adjusted to 3.4  Acetonitrile  d  0.02% formate with pH adjusted to 3.0  Acetonitrile  e  0.1% acetate acid with pH adjusted to 3.2  Acetonitrile  f  H2O  Acetonitrile with 0.05% acetic acid  Optimization of the gradient program Two different liquid chromatography system gradient modes were optimized. Chromatographic separation was achieved on a Phenomenex Gemini-NX C18 column (150 × 4.6 mm2, 3 μm). Solvent A was water and solvent B consisted of acetonitrile with 0.05% acetic acid. The one gradient started at 0% solvent B and increased to 60% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. The other linear gradient started at 10% solvent B and increased to 80% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. Chromatographic method validation and analysis of 4-fluoroaniline in ezetimibe API The LC–MS method developed above was validated for repeatability, linearity, accuracy, solution stability, robustness, limits of detection (LOD) and limits of quantification (LOQ). System suitability System suitability was tested by performing six consecutive injections using a concentration of standard mixture that was equivalent to the minimum quantifiable concentration (MQC) (15 ng/mL) of the calibration curve for 4-fluoroaniline. Precision The repeatability was evaluated with six experimental replicates of the same concentration, using a 15 ng/mL mixture of standards. Similarly, inter-day precision was assessed using the same samples the next day. Relative standard deviation (RSD) was used to evaluate repeatability, inter-day precision and robustness. Linearity Linearity was evaluated based on linear regression analyses of a calibration curve at concentrations equal to LOQ, as well as 5, 10, 15, 22.5 and 30 ng/mL. The peak areas for the same concentrations were averaged. A linear regression model was developed using the data, and a regression coefficient >0.9999 was considered indicative of a linear relationship between the peak area and the concentration of the 4-fluoroaniline. LOD and LOQ LOD was established using standards with concentrations of 0.19 ng/mL, and LOQ was established using standards with concentrations of 0.94 ng/mL. Results were obtained from six replicates. Accuracy The accuracy was assessed through the determination of 4-fluoroaniline recovery by adding different concentrations of 4-fluoroaniline standard solution to ezetimibe samples. The concentrations of 4-fluoroaniline standards added to ezetimibe samples were equal to LOQ, 15 and 18 ng/mL, with each assay performed separately. Solution stability The stability of 4-fluoroaniline standard solution was tested for 24 h at 25°C. The results were compared with the obtained at 0 h. Robustness The flow rate of the mobile phase was adjusted to ±10% of the desired value. The column temperature was changed by ±5°C. The pH value was changed by ±0.01%. In addition, the instrument was changed from the Agilent 6130 MSD mass spectrometer to the LCMS–Y mass spectrometer. Results Method development Under selected GC–MS conditions, the peak retention time for 4-fluoroaniline was ~5.7 min, which was relatively preferable with regard to 4-fluoroaniline standard. However, the recovery rate obtained using this method for 4-fluoroaniline was found to be above 120%. Ezetimibe was decomposed to 4-fluoroaniline under GC high-temperature conditions, which could be confirmed by the mass fragmentation mechanism of ezetimibe (22). According to test results obtained from the LC–MS method and through comparisons of resolution, tailing factor, and symmetry factor, the chromatographic column Phenomenex Gemini-NX C18 110 A (150 × 4.6 mm2, 3 μm) performed best. In the selected chromatographic column and gradient program, the composition of the mobile phase and its pH were optimized. First, aqueous ammonium formate and ammonium acetate were selected as buffer solution. Consequently, unsatisfactory chromatographic peaks and column efficiencies for 4-fluoroaniline using mobile phase cases a and b (Table I) were produced. The retention times of 4-fluoroaniline were nearly dead time. Then aqueous formate was chosen to optimize the mobile phase. For mobile phase cases c and d (Table I), tailing factor, and symmetry factor were not significantly improved when compared to results obtained from the use of aqueous ammonium formate and ammonium acetate. However, the retention time for 4-fluoroaniline was improved to some extent when pH of aqueous formate increased. Compared to formic acid (aqueous phase), the retention time was improved to approximately four times the dead time in acetate acid (aqueous phase) under the conditions of case e (Table I). However, changes in peak type, column efficiency and tailing factor were not significant, and a shoulder peak was observed in case e. So we had optimized the initial aqueous phase proportion for gradient elution. The initial aqueous phase proportion was changed to 90%. The acetic acid was mixed with the organic phase to reduce the acidity of the aqueous phase. Under the conditions of case f (Table I), the results indicate that the peak-to-peak tailing factor can be controlled to remain below 1.2 by reducing aqueous phase composition and acidity. LC–MS method validation Specificity Typical DAD chromatograms and MS output for the blank acetonitrile and mixed standard solutions of aniline, 4-fluoroaniline, o-fluoroaniline and 3-fluoroaniline (1.25 μg/mL) are shown in Figure 2. The results show that no interfering peaks were observed at the eluting position for 4-fluoroaniline. Figure 2. View largeDownload slide Typical DAD chromatograms and MS output for the mixed standard solution of aniline, 4-fluoroaniline, o-fluoroaniline and 3-fluoroaniline. The retention time of aniline, 4-fluoroaniline, 3-fluoroaniline, o-fluoroaniline and ezetimibe was 3.462, 4.074, 6.728, 6.784 and 9.638 min, respectively. Figure 2. View largeDownload slide Typical DAD chromatograms and MS output for the mixed standard solution of aniline, 4-fluoroaniline, o-fluoroaniline and 3-fluoroaniline. The retention time of aniline, 4-fluoroaniline, 3-fluoroaniline, o-fluoroaniline and ezetimibe was 3.462, 4.074, 6.728, 6.784 and 9.638 min, respectively. Calibration curve, LOQ and LOD Calibration standards, including 4-fluoroaniline solutions at concentrations of 0.94, 4.99, 9.99, 15.13, 22.70 and 30.26 ng/mL, were assayed. A typical equation of the calibration curve is as follows:   y=9216.9x+2526.2;(correlation coefficientr=0.9999,n=6) (1)Where, y is the peak-area ratio of 4-fluoroaniline, and x is the concentration of 4-fluoroaniline. LOQ was determined based on the minimum concentration of analyte that showed a linear peak area response. The LOQ for 4-fluoroaniline was 0.94 ng/mL. The LOD for 4-fluoroaniline was 0.19 ng/mL, which is sufficient to detect concentrations of the compound greater than the genotoxic impurity limit (0.015%) in ezetimibe. Precision and accuracy The intra-day (n = 6) and inter-day (n = 12) results are summarized in Table II. The intra-day and inter-day RSD were less than 8.7% (n = 12). The recovery of 4-fluoroaniline was between 92 and 99%. Thus, the data indicate that the present method has satisfactory precision and accuracy. Table II. Summary of Method Validation Item  Results  Sample Concentration  0.1 mg/mL  LOQ  0.94 ng/mL (equivalent to 0.00094% of sample concentration)  LOD  0.19 ng/mL (equivalent to 0.00019% of sample concentration)  Linearity  0.94–30.26 ng/mL, r = 0.9999  Recovery (mean ± SD)  94.19 ± 0.49% (LOQ), 99.59 ± 1.25% (15 ng/mL) 101.0 ± 1.56% (18 ng/mL)  Precisiona  8.68% (inter-day)  Item  Results  Sample Concentration  0.1 mg/mL  LOQ  0.94 ng/mL (equivalent to 0.00094% of sample concentration)  LOD  0.19 ng/mL (equivalent to 0.00019% of sample concentration)  Linearity  0.94–30.26 ng/mL, r = 0.9999  Recovery (mean ± SD)  94.19 ± 0.49% (LOQ), 99.59 ± 1.25% (15 ng/mL) 101.0 ± 1.56% (18 ng/mL)  Precisiona  8.68% (inter-day)  aBy RSD of the peak areas (n = 12). Table II. Summary of Method Validation Item  Results  Sample Concentration  0.1 mg/mL  LOQ  0.94 ng/mL (equivalent to 0.00094% of sample concentration)  LOD  0.19 ng/mL (equivalent to 0.00019% of sample concentration)  Linearity  0.94–30.26 ng/mL, r = 0.9999  Recovery (mean ± SD)  94.19 ± 0.49% (LOQ), 99.59 ± 1.25% (15 ng/mL) 101.0 ± 1.56% (18 ng/mL)  Precisiona  8.68% (inter-day)  Item  Results  Sample Concentration  0.1 mg/mL  LOQ  0.94 ng/mL (equivalent to 0.00094% of sample concentration)  LOD  0.19 ng/mL (equivalent to 0.00019% of sample concentration)  Linearity  0.94–30.26 ng/mL, r = 0.9999  Recovery (mean ± SD)  94.19 ± 0.49% (LOQ), 99.59 ± 1.25% (15 ng/mL) 101.0 ± 1.56% (18 ng/mL)  Precisiona  8.68% (inter-day)  aBy RSD of the peak areas (n = 12). Stability The stability of the 4-fluoroaniline standard solution was tested for 24 h at 25°C. High stability was observed under the tested condition. Robustness Changes in the mass spectrometer utilized, the pH value of the mobile phase, the flow rate, and the column temperature did not affect the determination of 4-fluoroaniline in ezetimibe. The method validation results are summarized in Tables II and III. Table III. The Robustness Results of 4-Fluoroaniline Analytical parameters  Parameter values  Added (ng/mL)  Found (ng/mL)  Recovery (%)  RSD (%)  Normal  /*  15.13  14.76  97.53  2.1  Acetic acid (%)  0.04  15.13  15.25  100.8  0.06  15.13  15.61  103.2  Flow rate (mL/min)  0.9  15.13  15.20  100.4  1.1  15.13  15.54  102.7  Oven column (°C)  25  15.13  14.97  98.95  35  15.13  15.12  99.96  Instrument  Agilent 6120 MSD  15.13  15.70  103.8  Analytical parameters  Parameter values  Added (ng/mL)  Found (ng/mL)  Recovery (%)  RSD (%)  Normal  /*  15.13  14.76  97.53  2.1  Acetic acid (%)  0.04  15.13  15.25  100.8  0.06  15.13  15.61  103.2  Flow rate (mL/min)  0.9  15.13  15.20  100.4  1.1  15.13  15.54  102.7  Oven column (°C)  25  15.13  14.97  98.95  35  15.13  15.12  99.96  Instrument  Agilent 6120 MSD  15.13  15.70  103.8  *See LC–MS system. Table III. The Robustness Results of 4-Fluoroaniline Analytical parameters  Parameter values  Added (ng/mL)  Found (ng/mL)  Recovery (%)  RSD (%)  Normal  /*  15.13  14.76  97.53  2.1  Acetic acid (%)  0.04  15.13  15.25  100.8  0.06  15.13  15.61  103.2  Flow rate (mL/min)  0.9  15.13  15.20  100.4  1.1  15.13  15.54  102.7  Oven column (°C)  25  15.13  14.97  98.95  35  15.13  15.12  99.96  Instrument  Agilent 6120 MSD  15.13  15.70  103.8  Analytical parameters  Parameter values  Added (ng/mL)  Found (ng/mL)  Recovery (%)  RSD (%)  Normal  /*  15.13  14.76  97.53  2.1  Acetic acid (%)  0.04  15.13  15.25  100.8  0.06  15.13  15.61  103.2  Flow rate (mL/min)  0.9  15.13  15.20  100.4  1.1  15.13  15.54  102.7  Oven column (°C)  25  15.13  14.97  98.95  35  15.13  15.12  99.96  Instrument  Agilent 6120 MSD  15.13  15.70  103.8  *See LC–MS system. Using the validated method, five batches of ezetimibe samples were analyzed. Approximately 0.001% of 4-fluoroaniline was detected in each batch, which is far below the TTC derived limit of 0.015% (Figure 3). Figure 3. View largeDownload slide Typical ezetimibe sample was analyzed using the validated method in MS and DAD detector. Approximately 0.001% 4-fluoroaniline was detected in each batch, which was far below the required limit of 0.015%. Figure 3. View largeDownload slide Typical ezetimibe sample was analyzed using the validated method in MS and DAD detector. Approximately 0.001% 4-fluoroaniline was detected in each batch, which was far below the required limit of 0.015%. Discussion Based on the physical and chemical properties of ezetimibe, it is easily decomposed to 4-fluoroaniline under high-temperature conditions (Figure 1) (22, 23). Therefore, the GC–MS method was not suitable for the detection of 4-fluoroaniline in ezetimibe. In view of the unsuitability of ezetimibe for measurement under GC conditions, we conducted studies on the residual 4-fluoroaniline in the drug by utilizing an LC–MS method, and optimized the method for its quantitative analysis. Since aniline compounds possess an amidogen structure, they are classified as weak-bases, which tend to protonate under assay conditions. Therefore, scanning in positive ion mode was selected for mass spectroscopy. Acetonitrile was chosen as the organic modifier because of its low interference and its equivalent peak shape. Acetic acid (0.05%) was required to achieve an acceptable peak shape and retention. A reverse phase Phenomenex Gemini-NX C18 column (150 mm × 4.6 mm i.d., 3 μ) with a mobile phase consisting of acetonitrile with 0.05% acetic acid (case f, Table I) in gradient mode was used in the finalized LC method. The retention time of 4-fluoroaniline was ~4 min. 4-Fluoroaniline is an aniline compound with potential genotoxicity. Strict limits have been placed on its residual amount in drugs. Calculation of the permissible limit of 4-fluoroaniline in each drug is carried out in accordance with the TTC value-based limit of 1.5 μg/day. The daily dose of ezetimibe is 10 mg, and the accepted residual limit of 4-fluoroaniline in this dose is 0.015%. High sensitivity and selectivity are required in any developed technique to control the quantity of a compound with such low allowable limits. The research method proposed in this paper meets these requirements. Our LC–MS technique combines the efficient separation capability of HPLC and the superior qualitative analysis capability of MS into one method, while also possessing the advantages of high speed, sensitivity and specificity (24). At high temperatures or in bright light, several drugs may be degraded into other molecular compounds, which are called degradation products (25). Some degradation products are similar in structure to the original substance, or already exist as components of the original substance. These degradation products may interfere with measurements. Since ezetimibe tends to decompose and produce 4-fluoroaniline under GC high-temperature and alkaline conditions (26), false positive results may occur. Moreover, other volatile components in the API tend to disrupt the detection of the substance of interest. This makes the GC–MS method unsuitable for the detection of components of heat-labile substances, such as ezetimibe. The LC–MS method applied in this article does not involve high-temperature conditions that result in the degradation of thermally unstable samples, which ensures both reliability and sensitivity of the detection. In the past, residual detection of aniline compounds focused on water quality, environmental pollution, and food contamination; there were few monitoring efforts related to genotoxic impurities, such as 4-fluoroaniline in drugs via the LC–MS method. Our study addresses this deficiency through its emphasis on the quantitative detection of 4-fluoroaniline in ezetimibe by LC–MS. As the detection can be carried out simply by dissolving the sample with diluents, the method is easy and simple to perform. Conclusions The present study reports a fully validated method that is suitable for the routine measurement of the very low concentrations of 4-fluoroaniline in ezetimibe. The method has high sensitivity, with an LOD of 0.19 ng/mL, and can be used for trace analysis of 4-fluoroaniline genotoxic impurities in ezetimibe. Furthermore, based on the physical and chemical properties of ezetimibe, we found that it is decomposed to 4-fluoroaniline under high-temperature conditions. Therefore, the GC–MS method is not suitable for the detection of 4-fluoroaniline impurities in ezetimibe. LC–MS can effectively prevent the degradation of ezetimibe to 4-fluoroaniline, a phenomenon that interferes with the accurate measurement of the genotoxic impurity. Sample preparation for LC–MS is simple and easy. This method is expected to help in determining and monitoring the levels of 4-fluoroaniline in thermally unstable drugs. Acknowledgment We thank Jiangsu Deyuan Pharmaceutical Co., Ltd. for providing us with ezetimibe API. References 1 Arrhenius, E., Hultin, T.; The effect of 2-aminofluorene and related aromatic amines on the protein and ribonucleic acid metabolism of liver slices; Experimental Cell Research , ( 1961); 22: 476– 486. Google Scholar CrossRef Search ADS PubMed  2 Scribner, J.D., Fisk, S.R., Scribner, N.K.; Mechanisms of action of carcinogenic aromatic amines: an investigation using mutagenesis in bacteria; Chemico-Biological Interactions , ( 1979); 26: 11– 25. Google Scholar CrossRef Search ADS PubMed  3 King, C.M., Land, S.J., Jones, R.F., Debiec-Rychter, M., Lee, M., Wang, C.Y.; Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines; Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis , ( 1997); 376: 123– 128. Google Scholar CrossRef Search ADS   4 Benigni, R., Passerini, L.; Carcinogenicity of the aromatic amines: from structure-activity relationships to mechanisms of action and risk assessment; Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis , ( 2002); 511: 191– 206. 5 Lakshmi, V.M., Hsu, F.F., Zenser, T.V.; Transformation and activation of benzidine by oxidants of the inflammatory response; Chemical Research in Toxicology , ( 2003); 16: 367– 374. Google Scholar CrossRef Search ADS PubMed  6 Hernando, D., Saurina, J., Cassou, S.H.; Liquid chromatographic determination of aniline in table-top sweeteners based on pre-column derivatization with 1,2-naphthoquinone-4-sulfonate; Journal of Chromatography A , ( 1999); 859: 227– 233. Google Scholar CrossRef Search ADS PubMed  7 Falciola, L., Pifferi, V., Mascheroni, E.; Platinum-based and carbon-based screen printed electrodes for the determination of benzidine by differential pulse voltammetry; Electroanalysis , ( 2012); 24: 767– 775. Google Scholar CrossRef Search ADS   8 Shuhui, L., Wenjun, W., Chen, J.; Determination of aniline and its derivatives in environmental water by capillary electrophoresis with on-line concentration; International Journal of Molecular Sciences , ( 2012); 13: 6863– 6872. Google Scholar CrossRef Search ADS PubMed  9 Chiang, J., Huang, S.; Simultaneous derivatization and extraction of anilines in waste water with dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometric detection; Talanta , ( 2007); 75: 70– 75. Google Scholar CrossRef Search ADS PubMed  10 Patel, G., Agrawal, Y.K.; Separation and trace estimation of benzidine and its macromolecular adducts using supercritical fluid chromatography; Journal of Chromatography B , ( 2003); 795: 157– 165. Google Scholar CrossRef Search ADS   11 Bundy, J.G., Lenz, E.M., Nicholson, J.K.; Metabolism of 4-fluoroaniline and 4-fluorobiphenyl in the earthworm Eisenia veneta characterized by high-resolution NMR spectroscopy with directly coupled HPLC–NMR and HPLC–MS; Xenobiotica; the Fate of Foreign Compounds in Biological Systems , ( 2002); 32: 479– 490. Google Scholar CrossRef Search ADS PubMed  12 Bundy, J.G., Lenz, E.M., Bailey, N.J., Gavaghan, C.L., Svendsen, C., Spurgeon, D., et al.  .; Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta (Rosa): identification of new endogenous biomarkers; Environmental Toxicology and Chemistry , ( 2002); 21: 1966– 1972. Google Scholar CrossRef Search ADS PubMed  13 Nakou, E.S., Filippatos, T.D., Kiortsis, D.N., Derdemezis, C.S., Tselepis, A.D., Mikhailidis, D.P., et al.  .; The effects of ezetimibe and orlistat, alone or in combination, on high-density lipoprotein (HDL) subclasses and HDL-associated enzyme activities in overweight and obese patients with hyperlipidaemia; Expert Opinion on Pharmacotherapy , ( 2008); 18: 3151– 3158. Google Scholar CrossRef Search ADS   14 Phan, B.A.P., Dayspring, T.D.; 2-Ezetimibe therapy: mechanism of action and clinical update; Vascular Health and Risk Management , ( 2012); 8: 415– 427. Google Scholar PubMed  15 Guideline, I.H.T. Q3A(R2): Impurities in new drug substance; ICH Steering Committee, ( 2006). 16 Guideline, I.H.T. Q3B(R2): Impurities in new drug products; ICH Steering Committee, ( 2006). 17 Guideline, I.H.T. Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; Geneva, ( 2014). 18 FDA. Guidance for Industry—genotoxic and carcinogenic impurities in drug substances and products: recommended approaches, ( 2008). 19 Douša, M., Klvaňa, R., Doubský, J., Srbek, J., Richter, J., Exner, M., et al.  .; HILIC–MS determination of genotoxic impurity of 2-chloro-N-(2-chloroethyl)ethanamine in the vortioxetine manufacturing process; Journal of Chromatographic Science , ( 2015); 54: 119– 124. Google Scholar PubMed  20 García, A., Rupérez, F.J., Ceppa, F., Pellati, F., Barbas, C.; Development of chromatographic methods for the determination of genotoxic impurities in cloperastine fendizoate; Journal of Chromatographic Science , ( 2012); 61: 230– 236. 21 National Standards of the People’s Republic of China. Water quality—determination of aniline compound–gas chromatography/mass spectrometry, ( 2014). 22 Raman, B., Sharma, B.A., Butala, R., Ghugare, P.D., Kumar, A.; Structural elucidation of a process-related impurity in ezetimibe by LC/MS/MS and NMR; Journal of Pharmaceutical and Biomedical Analysis , ( 2010); 52: 73– 78. Google Scholar CrossRef Search ADS PubMed  23 Guntupalli, S., Ray, U.K., Murali, N., Gupta, P.B., Kumar, V.J., Satheesh, D., et al.  .; Identification, isolation and characterization of process related impurities in ezetimibe; Journal of Pharmaceutical and Biomedical Analysis , ( 2014); 88: 385– 390. Google Scholar CrossRef Search ADS PubMed  24 Chen, X., Li, Y., Lin, X., Fan, Y., Huang, F., Jin, C.; Research progress on analysis on impurity in chemical drug by LC–MS technology; Drugs & Clinic , ( 2014); 29: 696– 700. 25 Pawale, S.S., Saley, S.P., Mundhada, D.R., Tilloo, S.K.; Impurity profile in bulk drugs and pharmaceutical preparation; International Journal of Pharmaceutical and Chemical Sciences , ( 2012); 1: 1227– 1237. 26 Gajjar, A.K., Shah, V.D.; Isolation and structure elucidation of major alkaline degradant of Ezetimibe; Journal of Pharmaceutical and Biomedical Analysis , ( 2011); 55: 225– 229. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Chromatographic Science Oxford University Press

Development of an LC–MS Method for 4-Fluoroaniline Determination in Ezetimibe

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
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0021-9665
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1945-239X
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10.1093/chromsci/bmy048
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Abstract

Abstract A rapid and sensitive high-performance liquid chromatography–mass spectrometry method was developed and validated to determine 4-fluoroaniline concentration in ezetimibe. Chromatographic separation was achieved on a Phenomenex Gemini-NX C18 column (150 × 4.6 mm, 3 μm) maintained at 30°C. The liquid chromatography system was operated in gradient mode with an injection volume of 20 μL at a flow rate of 1 mL/min. Mobile phase A was water and mobile phase B consisted of acetonitrile with 0.05% acetic acid. The detection was performed using a single quadrupole mass spectrometer in single ion monitoring mode by using positive ionization. An m/z value of 112 was selected for monitoring 4-fluoroaniline. The method showed good linearity over the concentration range of 0.94–30.26 ng/mL. The limit of quantification and limit of detection were 0.19 and 0.94 ng/mL, respectively. The precision relative standard deviations were less than 8.7% (n = 12), and the accuracy values were within 92–99%. A standard solution of 4-fluoroaniline was stable for at least 24 h at 25°C. Small changes in the organic phase acidity of the mobile phase, flow rate, column temperature, and the instrument parameters had no significant effect on the results for 4-fluoroaniline. Introduction The widespread use of aniline and its derivatives in the pesticide, plastic, and pharmaceutical industries has led to the contamination of aquatic environments and drug products with its chemical by-products. These compounds are well known due to their high toxicity and suspected carcinogenicity (1–4). Aniline compounds can be ingested, and this causes a decline in the oxygen-carrying capacity of hemoglobin, resulting in animal poisoning. In humans, aniline inhalation or absorption results in acute poisoning or chronic intoxication characterized by symptoms such as headaches, dizziness, drowsiness, memory loss, breathing difficulties and asphyxia. Long-term exposure to aniline compounds may induce cancers. The presence of residues of aniline in the environment has garnered attention that has resulted in its long-term inclusion on a list of contaminants requiring priority monitoring (5). Since anilines contain a benzene ring with a π conjugated system that can absorb ultraviolet (UV) radiation, UV determination and high-performance liquid chromatography (HPLC) can be employed for detection. Common techniques for the determination of aniline and its derivatives are spectrophotometry and chromatography. Initially, a diazo-coupling colorimetric method, using N-(1-naphthyl)ethylenediamine, was primarily used for the detection of aniline compounds through spectrophotometry at 545 nm. However, this method has relatively rigid requirements regarding the temperature of the chromogenic reaction and can produce unwanted reactions. However, most aniline compounds have low boiling points, and thus gas chromatography (GC) can be used to perform qualitative and quantitative studies. Although the GC method is easy to perform and has high specificity, it is not applicable to the study of compounds that produce aniline substances by high-temperature degradation. The constant evolution of science and technology has resulted in the emergence of more efficient and sensitive detection techniques that can be used to study residual amounts of aniline compounds. Some of these techniques are GC–tandem mass spectrometry (GC–MS-MS), liquid chromatography–tandem mass spectrometry (LC–MS-MS), liquid chromatography–nuclear magnetic resonance spectroscopy (LC–NMR), supercritical fluid chromatography (SFC), MS, electrochemical methods and nuclear magnetic resonance spectroscopy (NMR) (6–10). In 2002, Bundy et al. studied the metabolism of 4-fluoroaniline by Eisenia veneta and performed a quantitative study of its metabolic products by using HPLC coupled with 19F/1H–NMR (11, 12). Shuhui et al. established a capillary zone electrophoresis (CZE) with field-enhanced sample injection method for the direct determination of aniline and its derivatives in environmental water. The method was simple, sensitive, and environmentally friendly (8). In the aforementioned methods, little reference was made to the quantification of trace residual 4-fluoroaniline or determination of 4-fluoroaniline in thermally unstable drugs. 4-Fluoroaniline is a common fluorine-containing aromatic amine, widely used in the synthesis of medicines and pesticides, such as the drug ezetimibe (used to treat primary hypercholesterolemia), the drug norfloxacin (a broad-spectrum antibacterial agent), and a 4-amidogen quinoline pesticide. Ezetimibe is a selective inhibitor of intestinal cholesterol and phytosterol absorption, and is often used in combination with simvastatin, an HMG-CoA reductase inhibitor. Ezetimibe, the first specific inhibitor of the intestinal cholesterol uptake transporter Niemann-Pick C1-Like 1 (NPC1L1) protein, was developed as an agent to lower plasma levels of low-density lipoprotein cholesterol (LDL–C) (13, 14). 4-Fluoroaniline is used as a starting material in the synthesis of ezetimibe and can remain in the final product as an impurity (Figure 1). The International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) indicates that all compounds with aniline structures have potential genotoxicity, which warrants rigid control of their residual content in drugs. A threshold of toxicological concern (TTC) value of 1.5 μg of a mutagenic impurity per person per day is considered a negligible risk (theoretical excess cancer risk of <1 in 100,000 over a lifetime of exposure) and can be used for most pharmaceuticals as a default to derive an acceptable limit (15–20). From this calculation, considering the daily dose of ezetimibe to be ≥10 mg, the acceptable residual limit of 4-fluoroaniline is 0.015% (w/w). Thus far, there have been no studies on the determination of 4-fluoroaniline in drugs. This article reveals the findings of the first, thorough investigation of the small amounts of this genotoxic impurity, which is present in ezetimibe as a residual from the synthesis process, using a highly sensitive LC–MS method for detection. Figure 1. View largeDownload slide The synthesis and degradation processes of ezetimibe. Figure 1. View largeDownload slide The synthesis and degradation processes of ezetimibe. Materials and Methods Chemicals and materials Acetonitrile of LC–MS grade was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol of LC–MS grade was purchased from Merck (Darmstadt, Germany). Formic acid of analytical grade (97%) was obtained from Alfa Aesar (Tianjin, China). HPLC-grade trifluoroacetate was purchased from J&K Chemical Ltd. (Shanghai, China). Ammonium formate (≥99.999%) was purchased from Sigma-Aldrich. Ammonium acetate (≥98%) and glacial acetic acid (99.5%) were purchased from Sigma-Aldrich. Milli-Q water was of ultra-pure quality (>18 MΩ/cm) and prepared in-house. Nitrogen (99.999%) was purchased from Shengtang (Tianjin, China). 3-Fluoroaniline, 4-fluoroaniline and aniline were all of >95% purity and were purchased from Alfa Aesar (Tianjin, China). Ezetimibe was provided as a gift sample by DEYUAN Pharmaceutical Ltd. (Lianyungang, China). Instrumentation GC–MS system The GC–MS system consisted of a Shimadzu GCMS-QP2010 Plus gas chromatography–mass spectrometer and Agilent 7694E headspace gas chromatography injection system. The system was operated using the Shimadzu GC–MS solution software operation system. The chromatography was performed using a 0.25 μm, 30 m × 0.25 mm (i.d.) capillary column (DB-5MS). The carrier gas was nitrogen, and the flow rate was 1 mL/min (constant). The oven temperature was initially 60°C and was kept at this level for 5 min. It was then increased to 140°C at the rate of 10°C/min, kept constant for 1 min, subsequently increased to 220°C at the rate of 30°C/min, and finally kept steady for 1 min. The inlet temperature was 250°C. The ion source temperature was 230°C. The mass spectrometer used for analysis was equipped with an EI source and data acquisition was carried out in single ion monitoring (SIM) mode. The following conditions were used in the headspace instrument: the injection volume was 1 mL using headspace injection mode with a split made in a 10:1 ratio, the headspace equilibrium temperature was 110°C, loop temperature was 120°C, and transfer line temperature was 130°C. The total run time of the method was 7.4 min. LC–MS system The LC–MS system consisted of an Agilent 1260 with a G1379B 1260u-Degasser, G1312B 1260 Bin Pump system, G1316C 1260 TCC column oven and a G4212B 1260 DAD detector connected to an Agilent 6130 MSD mass spectrometer. The system was operated using Agilent ChemStation software. The chromatography was performed using a 3 μm 110 A, 150 × 4.6 mm (i.d.) C18 column (Phenomenex Gemini-NX). The liquid chromatography system was operated in a gradient mode with a flow rate of 1 mL/min. Solvent A was water and solvent B consisted of acetonitrile with 0.05% acetic acid. The linear gradient started at 10% solvent B and increased to 80% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. Starting conditions were achieved in 1 min and the column was re-equilibrated for 5 min. The injection volume was 20 μL and the column oven temperature was 30°C. Needle wash was performed before and after injection. The total run time was 15 min. In the mass spectrometer, positive electrospray ionization (ESI) was used. Data acquisition was carried out in SIM mode using positive ionization; nitrogen was used as sheath, auxiliary and ion sweep gas. Applied spray pressure was 35 psi, drying gas flow was 10 mL/min, drying gas temperature was 350°C, and fragment was 80 V. Preparation of standards and samples GC–MS Standard stock solutions of 4-fluoroaniline were prepared in methanol at a final concentration of 1.5 mg/mL. The 4-fluoroaniline stock solution was diluted in methanol to achieve a final concentration of 15 ng/mL. Sample solutions of ezetimibe were prepared in methanol at final concentrations of 0.1 mg/mL, and were injected into the GC–MS system after filtration through 0.45 μm nylon filters. LC–MS Standard stock solutions of 4-fluoroaniline were prepared in acetonitrile at a final concentration of 1.5 mg/mL. These solutions were stored at 2–8°C until use. The 4-fluoroaniline stock solution was diluted in acetonitrile to achieve a final concentration of 15 ng/mL. Standards of 4-fluoroaniline, to be used for the preparation of calibration curves, were prepared in acetonitrile by serial dilution over the concentration range 0.94–30 ng/mL. Mixed standard solutions of 4-fluoroaniline, o-fluoroaniline, 3-fluoroaniline and aniline were prepared in acetonitrile at a final concentration of 15 ng/mL. Sample solutions of ezetimibe were prepared in acetonitrile at a final concentration of 0.1 mg/mL and injected into the LC–MS system after filtration through 0.45 μm nylon filters. Development and optimization of chromatographic analysis GC–MS Possible residual 4-fluoroaniline in ezetimibe was detected by applying the GC–MS method according to published protocols, as well as information regarding the physical and chemical properties of 4-fluoroaniline (21). The detailed GC–MS conditions were given in GC–MS system. LC–MS Optimization of the column One milliliter of mixed standard solution was transferred to a flask, and used to test individual chromatographic columns with different specifications. Examples included Phenomenex Gemini-NX C18 110 A (150 × 4.6 mm2, 3 μm), Phenomenex Kinetex C18 (150 × 4.6 mm2, 2.5 μm), Waters Sunfire C18, (150 × 4.6 mm2, 3.5 μm), and Waters XBridge C18, (150 × 4.6 mm2, 5 μm). The liquid chromatography system was operated in a gradient mode with a flow rate of 1 mL/min. Solvent A was water and solvent B consisted of acetonitrile with 0.05% acetic acid. The linear gradient started at 0% solvent B and increased to 60% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. Optimization of the mobile phase Six different mobile phases (cases a–f, Table I) were selected and tested to optimize the composition of the mobile phase. Chromatographic separation was achieved on a Phenomenex Gemini-NX C18 column (150 × 4.6 mm2, 3 μm) maintained at 30°C, following a gradient program. In each case, the linear gradient started at 0% solvent B and increased to 60% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. Table I. Mobile Phase Composition Used in the LC–MS Method Development Case  Mobile phase composition  A  B  a  10 mM ammonium formate with pH adjusted to 4.5  Acetonitrile  b  10 mM ammonium acetate with pH adjusted to 6.7  Acetonitrile  c  0.004% formate with pH adjusted to 3.4  Acetonitrile  d  0.02% formate with pH adjusted to 3.0  Acetonitrile  e  0.1% acetate acid with pH adjusted to 3.2  Acetonitrile  f  H2O  Acetonitrile with 0.05% acetic acid  Case  Mobile phase composition  A  B  a  10 mM ammonium formate with pH adjusted to 4.5  Acetonitrile  b  10 mM ammonium acetate with pH adjusted to 6.7  Acetonitrile  c  0.004% formate with pH adjusted to 3.4  Acetonitrile  d  0.02% formate with pH adjusted to 3.0  Acetonitrile  e  0.1% acetate acid with pH adjusted to 3.2  Acetonitrile  f  H2O  Acetonitrile with 0.05% acetic acid  Table I. Mobile Phase Composition Used in the LC–MS Method Development Case  Mobile phase composition  A  B  a  10 mM ammonium formate with pH adjusted to 4.5  Acetonitrile  b  10 mM ammonium acetate with pH adjusted to 6.7  Acetonitrile  c  0.004% formate with pH adjusted to 3.4  Acetonitrile  d  0.02% formate with pH adjusted to 3.0  Acetonitrile  e  0.1% acetate acid with pH adjusted to 3.2  Acetonitrile  f  H2O  Acetonitrile with 0.05% acetic acid  Case  Mobile phase composition  A  B  a  10 mM ammonium formate with pH adjusted to 4.5  Acetonitrile  b  10 mM ammonium acetate with pH adjusted to 6.7  Acetonitrile  c  0.004% formate with pH adjusted to 3.4  Acetonitrile  d  0.02% formate with pH adjusted to 3.0  Acetonitrile  e  0.1% acetate acid with pH adjusted to 3.2  Acetonitrile  f  H2O  Acetonitrile with 0.05% acetic acid  Optimization of the gradient program Two different liquid chromatography system gradient modes were optimized. Chromatographic separation was achieved on a Phenomenex Gemini-NX C18 column (150 × 4.6 mm2, 3 μm). Solvent A was water and solvent B consisted of acetonitrile with 0.05% acetic acid. The one gradient started at 0% solvent B and increased to 60% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. The other linear gradient started at 10% solvent B and increased to 80% solvent B within 10 min. In the following 5 min, solvent B percentage was increased to 100%. Chromatographic method validation and analysis of 4-fluoroaniline in ezetimibe API The LC–MS method developed above was validated for repeatability, linearity, accuracy, solution stability, robustness, limits of detection (LOD) and limits of quantification (LOQ). System suitability System suitability was tested by performing six consecutive injections using a concentration of standard mixture that was equivalent to the minimum quantifiable concentration (MQC) (15 ng/mL) of the calibration curve for 4-fluoroaniline. Precision The repeatability was evaluated with six experimental replicates of the same concentration, using a 15 ng/mL mixture of standards. Similarly, inter-day precision was assessed using the same samples the next day. Relative standard deviation (RSD) was used to evaluate repeatability, inter-day precision and robustness. Linearity Linearity was evaluated based on linear regression analyses of a calibration curve at concentrations equal to LOQ, as well as 5, 10, 15, 22.5 and 30 ng/mL. The peak areas for the same concentrations were averaged. A linear regression model was developed using the data, and a regression coefficient >0.9999 was considered indicative of a linear relationship between the peak area and the concentration of the 4-fluoroaniline. LOD and LOQ LOD was established using standards with concentrations of 0.19 ng/mL, and LOQ was established using standards with concentrations of 0.94 ng/mL. Results were obtained from six replicates. Accuracy The accuracy was assessed through the determination of 4-fluoroaniline recovery by adding different concentrations of 4-fluoroaniline standard solution to ezetimibe samples. The concentrations of 4-fluoroaniline standards added to ezetimibe samples were equal to LOQ, 15 and 18 ng/mL, with each assay performed separately. Solution stability The stability of 4-fluoroaniline standard solution was tested for 24 h at 25°C. The results were compared with the obtained at 0 h. Robustness The flow rate of the mobile phase was adjusted to ±10% of the desired value. The column temperature was changed by ±5°C. The pH value was changed by ±0.01%. In addition, the instrument was changed from the Agilent 6130 MSD mass spectrometer to the LCMS–Y mass spectrometer. Results Method development Under selected GC–MS conditions, the peak retention time for 4-fluoroaniline was ~5.7 min, which was relatively preferable with regard to 4-fluoroaniline standard. However, the recovery rate obtained using this method for 4-fluoroaniline was found to be above 120%. Ezetimibe was decomposed to 4-fluoroaniline under GC high-temperature conditions, which could be confirmed by the mass fragmentation mechanism of ezetimibe (22). According to test results obtained from the LC–MS method and through comparisons of resolution, tailing factor, and symmetry factor, the chromatographic column Phenomenex Gemini-NX C18 110 A (150 × 4.6 mm2, 3 μm) performed best. In the selected chromatographic column and gradient program, the composition of the mobile phase and its pH were optimized. First, aqueous ammonium formate and ammonium acetate were selected as buffer solution. Consequently, unsatisfactory chromatographic peaks and column efficiencies for 4-fluoroaniline using mobile phase cases a and b (Table I) were produced. The retention times of 4-fluoroaniline were nearly dead time. Then aqueous formate was chosen to optimize the mobile phase. For mobile phase cases c and d (Table I), tailing factor, and symmetry factor were not significantly improved when compared to results obtained from the use of aqueous ammonium formate and ammonium acetate. However, the retention time for 4-fluoroaniline was improved to some extent when pH of aqueous formate increased. Compared to formic acid (aqueous phase), the retention time was improved to approximately four times the dead time in acetate acid (aqueous phase) under the conditions of case e (Table I). However, changes in peak type, column efficiency and tailing factor were not significant, and a shoulder peak was observed in case e. So we had optimized the initial aqueous phase proportion for gradient elution. The initial aqueous phase proportion was changed to 90%. The acetic acid was mixed with the organic phase to reduce the acidity of the aqueous phase. Under the conditions of case f (Table I), the results indicate that the peak-to-peak tailing factor can be controlled to remain below 1.2 by reducing aqueous phase composition and acidity. LC–MS method validation Specificity Typical DAD chromatograms and MS output for the blank acetonitrile and mixed standard solutions of aniline, 4-fluoroaniline, o-fluoroaniline and 3-fluoroaniline (1.25 μg/mL) are shown in Figure 2. The results show that no interfering peaks were observed at the eluting position for 4-fluoroaniline. Figure 2. View largeDownload slide Typical DAD chromatograms and MS output for the mixed standard solution of aniline, 4-fluoroaniline, o-fluoroaniline and 3-fluoroaniline. The retention time of aniline, 4-fluoroaniline, 3-fluoroaniline, o-fluoroaniline and ezetimibe was 3.462, 4.074, 6.728, 6.784 and 9.638 min, respectively. Figure 2. View largeDownload slide Typical DAD chromatograms and MS output for the mixed standard solution of aniline, 4-fluoroaniline, o-fluoroaniline and 3-fluoroaniline. The retention time of aniline, 4-fluoroaniline, 3-fluoroaniline, o-fluoroaniline and ezetimibe was 3.462, 4.074, 6.728, 6.784 and 9.638 min, respectively. Calibration curve, LOQ and LOD Calibration standards, including 4-fluoroaniline solutions at concentrations of 0.94, 4.99, 9.99, 15.13, 22.70 and 30.26 ng/mL, were assayed. A typical equation of the calibration curve is as follows:   y=9216.9x+2526.2;(correlation coefficientr=0.9999,n=6) (1)Where, y is the peak-area ratio of 4-fluoroaniline, and x is the concentration of 4-fluoroaniline. LOQ was determined based on the minimum concentration of analyte that showed a linear peak area response. The LOQ for 4-fluoroaniline was 0.94 ng/mL. The LOD for 4-fluoroaniline was 0.19 ng/mL, which is sufficient to detect concentrations of the compound greater than the genotoxic impurity limit (0.015%) in ezetimibe. Precision and accuracy The intra-day (n = 6) and inter-day (n = 12) results are summarized in Table II. The intra-day and inter-day RSD were less than 8.7% (n = 12). The recovery of 4-fluoroaniline was between 92 and 99%. Thus, the data indicate that the present method has satisfactory precision and accuracy. Table II. Summary of Method Validation Item  Results  Sample Concentration  0.1 mg/mL  LOQ  0.94 ng/mL (equivalent to 0.00094% of sample concentration)  LOD  0.19 ng/mL (equivalent to 0.00019% of sample concentration)  Linearity  0.94–30.26 ng/mL, r = 0.9999  Recovery (mean ± SD)  94.19 ± 0.49% (LOQ), 99.59 ± 1.25% (15 ng/mL) 101.0 ± 1.56% (18 ng/mL)  Precisiona  8.68% (inter-day)  Item  Results  Sample Concentration  0.1 mg/mL  LOQ  0.94 ng/mL (equivalent to 0.00094% of sample concentration)  LOD  0.19 ng/mL (equivalent to 0.00019% of sample concentration)  Linearity  0.94–30.26 ng/mL, r = 0.9999  Recovery (mean ± SD)  94.19 ± 0.49% (LOQ), 99.59 ± 1.25% (15 ng/mL) 101.0 ± 1.56% (18 ng/mL)  Precisiona  8.68% (inter-day)  aBy RSD of the peak areas (n = 12). Table II. Summary of Method Validation Item  Results  Sample Concentration  0.1 mg/mL  LOQ  0.94 ng/mL (equivalent to 0.00094% of sample concentration)  LOD  0.19 ng/mL (equivalent to 0.00019% of sample concentration)  Linearity  0.94–30.26 ng/mL, r = 0.9999  Recovery (mean ± SD)  94.19 ± 0.49% (LOQ), 99.59 ± 1.25% (15 ng/mL) 101.0 ± 1.56% (18 ng/mL)  Precisiona  8.68% (inter-day)  Item  Results  Sample Concentration  0.1 mg/mL  LOQ  0.94 ng/mL (equivalent to 0.00094% of sample concentration)  LOD  0.19 ng/mL (equivalent to 0.00019% of sample concentration)  Linearity  0.94–30.26 ng/mL, r = 0.9999  Recovery (mean ± SD)  94.19 ± 0.49% (LOQ), 99.59 ± 1.25% (15 ng/mL) 101.0 ± 1.56% (18 ng/mL)  Precisiona  8.68% (inter-day)  aBy RSD of the peak areas (n = 12). Stability The stability of the 4-fluoroaniline standard solution was tested for 24 h at 25°C. High stability was observed under the tested condition. Robustness Changes in the mass spectrometer utilized, the pH value of the mobile phase, the flow rate, and the column temperature did not affect the determination of 4-fluoroaniline in ezetimibe. The method validation results are summarized in Tables II and III. Table III. The Robustness Results of 4-Fluoroaniline Analytical parameters  Parameter values  Added (ng/mL)  Found (ng/mL)  Recovery (%)  RSD (%)  Normal  /*  15.13  14.76  97.53  2.1  Acetic acid (%)  0.04  15.13  15.25  100.8  0.06  15.13  15.61  103.2  Flow rate (mL/min)  0.9  15.13  15.20  100.4  1.1  15.13  15.54  102.7  Oven column (°C)  25  15.13  14.97  98.95  35  15.13  15.12  99.96  Instrument  Agilent 6120 MSD  15.13  15.70  103.8  Analytical parameters  Parameter values  Added (ng/mL)  Found (ng/mL)  Recovery (%)  RSD (%)  Normal  /*  15.13  14.76  97.53  2.1  Acetic acid (%)  0.04  15.13  15.25  100.8  0.06  15.13  15.61  103.2  Flow rate (mL/min)  0.9  15.13  15.20  100.4  1.1  15.13  15.54  102.7  Oven column (°C)  25  15.13  14.97  98.95  35  15.13  15.12  99.96  Instrument  Agilent 6120 MSD  15.13  15.70  103.8  *See LC–MS system. Table III. The Robustness Results of 4-Fluoroaniline Analytical parameters  Parameter values  Added (ng/mL)  Found (ng/mL)  Recovery (%)  RSD (%)  Normal  /*  15.13  14.76  97.53  2.1  Acetic acid (%)  0.04  15.13  15.25  100.8  0.06  15.13  15.61  103.2  Flow rate (mL/min)  0.9  15.13  15.20  100.4  1.1  15.13  15.54  102.7  Oven column (°C)  25  15.13  14.97  98.95  35  15.13  15.12  99.96  Instrument  Agilent 6120 MSD  15.13  15.70  103.8  Analytical parameters  Parameter values  Added (ng/mL)  Found (ng/mL)  Recovery (%)  RSD (%)  Normal  /*  15.13  14.76  97.53  2.1  Acetic acid (%)  0.04  15.13  15.25  100.8  0.06  15.13  15.61  103.2  Flow rate (mL/min)  0.9  15.13  15.20  100.4  1.1  15.13  15.54  102.7  Oven column (°C)  25  15.13  14.97  98.95  35  15.13  15.12  99.96  Instrument  Agilent 6120 MSD  15.13  15.70  103.8  *See LC–MS system. Using the validated method, five batches of ezetimibe samples were analyzed. Approximately 0.001% of 4-fluoroaniline was detected in each batch, which is far below the TTC derived limit of 0.015% (Figure 3). Figure 3. View largeDownload slide Typical ezetimibe sample was analyzed using the validated method in MS and DAD detector. Approximately 0.001% 4-fluoroaniline was detected in each batch, which was far below the required limit of 0.015%. Figure 3. View largeDownload slide Typical ezetimibe sample was analyzed using the validated method in MS and DAD detector. Approximately 0.001% 4-fluoroaniline was detected in each batch, which was far below the required limit of 0.015%. Discussion Based on the physical and chemical properties of ezetimibe, it is easily decomposed to 4-fluoroaniline under high-temperature conditions (Figure 1) (22, 23). Therefore, the GC–MS method was not suitable for the detection of 4-fluoroaniline in ezetimibe. In view of the unsuitability of ezetimibe for measurement under GC conditions, we conducted studies on the residual 4-fluoroaniline in the drug by utilizing an LC–MS method, and optimized the method for its quantitative analysis. Since aniline compounds possess an amidogen structure, they are classified as weak-bases, which tend to protonate under assay conditions. Therefore, scanning in positive ion mode was selected for mass spectroscopy. Acetonitrile was chosen as the organic modifier because of its low interference and its equivalent peak shape. Acetic acid (0.05%) was required to achieve an acceptable peak shape and retention. A reverse phase Phenomenex Gemini-NX C18 column (150 mm × 4.6 mm i.d., 3 μ) with a mobile phase consisting of acetonitrile with 0.05% acetic acid (case f, Table I) in gradient mode was used in the finalized LC method. The retention time of 4-fluoroaniline was ~4 min. 4-Fluoroaniline is an aniline compound with potential genotoxicity. Strict limits have been placed on its residual amount in drugs. Calculation of the permissible limit of 4-fluoroaniline in each drug is carried out in accordance with the TTC value-based limit of 1.5 μg/day. The daily dose of ezetimibe is 10 mg, and the accepted residual limit of 4-fluoroaniline in this dose is 0.015%. High sensitivity and selectivity are required in any developed technique to control the quantity of a compound with such low allowable limits. The research method proposed in this paper meets these requirements. Our LC–MS technique combines the efficient separation capability of HPLC and the superior qualitative analysis capability of MS into one method, while also possessing the advantages of high speed, sensitivity and specificity (24). At high temperatures or in bright light, several drugs may be degraded into other molecular compounds, which are called degradation products (25). Some degradation products are similar in structure to the original substance, or already exist as components of the original substance. These degradation products may interfere with measurements. Since ezetimibe tends to decompose and produce 4-fluoroaniline under GC high-temperature and alkaline conditions (26), false positive results may occur. Moreover, other volatile components in the API tend to disrupt the detection of the substance of interest. This makes the GC–MS method unsuitable for the detection of components of heat-labile substances, such as ezetimibe. The LC–MS method applied in this article does not involve high-temperature conditions that result in the degradation of thermally unstable samples, which ensures both reliability and sensitivity of the detection. In the past, residual detection of aniline compounds focused on water quality, environmental pollution, and food contamination; there were few monitoring efforts related to genotoxic impurities, such as 4-fluoroaniline in drugs via the LC–MS method. Our study addresses this deficiency through its emphasis on the quantitative detection of 4-fluoroaniline in ezetimibe by LC–MS. As the detection can be carried out simply by dissolving the sample with diluents, the method is easy and simple to perform. Conclusions The present study reports a fully validated method that is suitable for the routine measurement of the very low concentrations of 4-fluoroaniline in ezetimibe. The method has high sensitivity, with an LOD of 0.19 ng/mL, and can be used for trace analysis of 4-fluoroaniline genotoxic impurities in ezetimibe. Furthermore, based on the physical and chemical properties of ezetimibe, we found that it is decomposed to 4-fluoroaniline under high-temperature conditions. Therefore, the GC–MS method is not suitable for the detection of 4-fluoroaniline impurities in ezetimibe. LC–MS can effectively prevent the degradation of ezetimibe to 4-fluoroaniline, a phenomenon that interferes with the accurate measurement of the genotoxic impurity. Sample preparation for LC–MS is simple and easy. This method is expected to help in determining and monitoring the levels of 4-fluoroaniline in thermally unstable drugs. Acknowledgment We thank Jiangsu Deyuan Pharmaceutical Co., Ltd. for providing us with ezetimibe API. References 1 Arrhenius, E., Hultin, T.; The effect of 2-aminofluorene and related aromatic amines on the protein and ribonucleic acid metabolism of liver slices; Experimental Cell Research , ( 1961); 22: 476– 486. Google Scholar CrossRef Search ADS PubMed  2 Scribner, J.D., Fisk, S.R., Scribner, N.K.; Mechanisms of action of carcinogenic aromatic amines: an investigation using mutagenesis in bacteria; Chemico-Biological Interactions , ( 1979); 26: 11– 25. Google Scholar CrossRef Search ADS PubMed  3 King, C.M., Land, S.J., Jones, R.F., Debiec-Rychter, M., Lee, M., Wang, C.Y.; Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines; Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis , ( 1997); 376: 123– 128. Google Scholar CrossRef Search ADS   4 Benigni, R., Passerini, L.; Carcinogenicity of the aromatic amines: from structure-activity relationships to mechanisms of action and risk assessment; Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis , ( 2002); 511: 191– 206. 5 Lakshmi, V.M., Hsu, F.F., Zenser, T.V.; Transformation and activation of benzidine by oxidants of the inflammatory response; Chemical Research in Toxicology , ( 2003); 16: 367– 374. Google Scholar CrossRef Search ADS PubMed  6 Hernando, D., Saurina, J., Cassou, S.H.; Liquid chromatographic determination of aniline in table-top sweeteners based on pre-column derivatization with 1,2-naphthoquinone-4-sulfonate; Journal of Chromatography A , ( 1999); 859: 227– 233. Google Scholar CrossRef Search ADS PubMed  7 Falciola, L., Pifferi, V., Mascheroni, E.; Platinum-based and carbon-based screen printed electrodes for the determination of benzidine by differential pulse voltammetry; Electroanalysis , ( 2012); 24: 767– 775. Google Scholar CrossRef Search ADS   8 Shuhui, L., Wenjun, W., Chen, J.; Determination of aniline and its derivatives in environmental water by capillary electrophoresis with on-line concentration; International Journal of Molecular Sciences , ( 2012); 13: 6863– 6872. Google Scholar CrossRef Search ADS PubMed  9 Chiang, J., Huang, S.; Simultaneous derivatization and extraction of anilines in waste water with dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometric detection; Talanta , ( 2007); 75: 70– 75. Google Scholar CrossRef Search ADS PubMed  10 Patel, G., Agrawal, Y.K.; Separation and trace estimation of benzidine and its macromolecular adducts using supercritical fluid chromatography; Journal of Chromatography B , ( 2003); 795: 157– 165. Google Scholar CrossRef Search ADS   11 Bundy, J.G., Lenz, E.M., Nicholson, J.K.; Metabolism of 4-fluoroaniline and 4-fluorobiphenyl in the earthworm Eisenia veneta characterized by high-resolution NMR spectroscopy with directly coupled HPLC–NMR and HPLC–MS; Xenobiotica; the Fate of Foreign Compounds in Biological Systems , ( 2002); 32: 479– 490. Google Scholar CrossRef Search ADS PubMed  12 Bundy, J.G., Lenz, E.M., Bailey, N.J., Gavaghan, C.L., Svendsen, C., Spurgeon, D., et al.  .; Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta (Rosa): identification of new endogenous biomarkers; Environmental Toxicology and Chemistry , ( 2002); 21: 1966– 1972. Google Scholar CrossRef Search ADS PubMed  13 Nakou, E.S., Filippatos, T.D., Kiortsis, D.N., Derdemezis, C.S., Tselepis, A.D., Mikhailidis, D.P., et al.  .; The effects of ezetimibe and orlistat, alone or in combination, on high-density lipoprotein (HDL) subclasses and HDL-associated enzyme activities in overweight and obese patients with hyperlipidaemia; Expert Opinion on Pharmacotherapy , ( 2008); 18: 3151– 3158. Google Scholar CrossRef Search ADS   14 Phan, B.A.P., Dayspring, T.D.; 2-Ezetimibe therapy: mechanism of action and clinical update; Vascular Health and Risk Management , ( 2012); 8: 415– 427. Google Scholar PubMed  15 Guideline, I.H.T. Q3A(R2): Impurities in new drug substance; ICH Steering Committee, ( 2006). 16 Guideline, I.H.T. Q3B(R2): Impurities in new drug products; ICH Steering Committee, ( 2006). 17 Guideline, I.H.T. Assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; Geneva, ( 2014). 18 FDA. Guidance for Industry—genotoxic and carcinogenic impurities in drug substances and products: recommended approaches, ( 2008). 19 Douša, M., Klvaňa, R., Doubský, J., Srbek, J., Richter, J., Exner, M., et al.  .; HILIC–MS determination of genotoxic impurity of 2-chloro-N-(2-chloroethyl)ethanamine in the vortioxetine manufacturing process; Journal of Chromatographic Science , ( 2015); 54: 119– 124. Google Scholar PubMed  20 García, A., Rupérez, F.J., Ceppa, F., Pellati, F., Barbas, C.; Development of chromatographic methods for the determination of genotoxic impurities in cloperastine fendizoate; Journal of Chromatographic Science , ( 2012); 61: 230– 236. 21 National Standards of the People’s Republic of China. Water quality—determination of aniline compound–gas chromatography/mass spectrometry, ( 2014). 22 Raman, B., Sharma, B.A., Butala, R., Ghugare, P.D., Kumar, A.; Structural elucidation of a process-related impurity in ezetimibe by LC/MS/MS and NMR; Journal of Pharmaceutical and Biomedical Analysis , ( 2010); 52: 73– 78. Google Scholar CrossRef Search ADS PubMed  23 Guntupalli, S., Ray, U.K., Murali, N., Gupta, P.B., Kumar, V.J., Satheesh, D., et al.  .; Identification, isolation and characterization of process related impurities in ezetimibe; Journal of Pharmaceutical and Biomedical Analysis , ( 2014); 88: 385– 390. Google Scholar CrossRef Search ADS PubMed  24 Chen, X., Li, Y., Lin, X., Fan, Y., Huang, F., Jin, C.; Research progress on analysis on impurity in chemical drug by LC–MS technology; Drugs & Clinic , ( 2014); 29: 696– 700. 25 Pawale, S.S., Saley, S.P., Mundhada, D.R., Tilloo, S.K.; Impurity profile in bulk drugs and pharmaceutical preparation; International Journal of Pharmaceutical and Chemical Sciences , ( 2012); 1: 1227– 1237. 26 Gajjar, A.K., Shah, V.D.; Isolation and structure elucidation of major alkaline degradant of Ezetimibe; Journal of Pharmaceutical and Biomedical Analysis , ( 2011); 55: 225– 229. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Journal of Chromatographic ScienceOxford University Press

Published: May 14, 2018

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