Validation of chromatographic method for impurity profiling of Baloxavir marboxil (Xofluza)

2.1 Instrumentations and chemicals

Diane liquid chromatograph U3000 (UV detector), ZORBAX SB-Phenyl C18 column (5 µm, 4.6 × 250 mm), KNAUER-AZURA HPLC system (Knauer, Germany), Ceres B preparation column (7 μm, 250 × 30 mm); Bruker AVIII 400 MHz nuclear magnetic resonance (NMR) spectrometer; Mettler TOLEDO-XSE205DU electronic balance.

Water was purified using the Milli-Q water purification system (Millipore, USA); BXM intermediate (Shandong Xinhua Pharmaceutical Co., Ltd. Zibo, China); and chromatographic methanol and analytical grade phosphoric acid were acquired from Merck (Darmstadt, Germany).

2.2 Procedure

The intermediate I reference substance (5 mg) was dissolved in methanol before being adjusted in volume (10 mL) and evenly shaken to obtain the reference substance reserve solution. The stock solutions were diluted to achieve various concentrations of reference working solutions. Furthermore, the same procedure was repeated for other intermediates. BXM intermediates are indicated in Table 1.

Table 1

BXM Intermediates

BXM intermediates	Structure	IUPAC names
Starting material II		3,4-Difluoro-2-methylbenzoic acid
Intermediate III		Methyl 3,4-difluoro-2-methylbenzoate
Intermediate IV		Methyl 3,4-difluoro-2-bromomethyl benzoate
Intermediate V		Methyl 3,4-difluoro-2-((phenylthio)methyl)benzoate
Intermediate VI		3,4-Difluoro-2-((phenylthio)methyl)benzoic acid
Intermediate VII		7,8-Difluoro-dibenzo[b,e]thiepin-11(6H)-one
Intermediate I		7,8-Difluoro-6,11-dihydro-dibenzo[b,e]thiepin-11-ol
Intermediate VIII		(12aR)-12-[(11S)-7,8-Difluoro-6,11-dihydrodibenzo[b,e] thiepin-11-yl]-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-benzyloxy
Intermediate IX (Baloxavir acid)		(12aR)-12-[(11S)-7,8-Difluoro-6,11-dihydrodibenzo[b,e] thiepin-11-yl]-6,8-dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-ol
BXM		({(12aR)-12-[(11S)-7,8-Difluoro-6,11-dihydrodibenzo[b,e] thiepin11-yl]-6,8- dioxo-3,4,6,8,12,12a-hexahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy)methylmethyl carbonate

Next, gradient elution was carried out using the following parameters: flow rate = 1.0 mL/min; sample volume = 10 μL; detection wavelength = 210 nm; column temperature = 30°C; mobile phase A = 0.1% phosphoric acid solution with pH = 4.0; and mobile phase B = methanol, as shown in Table 2.

2.3 Preparation and separation

To acquire a test sample solution, 0.4 g of intermediate A1 was dissolved in 8 mL of water and acetonitrile using ultrasonic waves, followed by filtration through an organic membrane (0.45 μm). The flow rate was 30 mL/min, the UV wavelength was 268 nm, and the mobile phase consisted of 30/70 (v/v) acetonitrile/water.

2.4 Characterization and analysis of impurities

The Agilent 6520 Accurate-Mass Q-TOF (USA) performed ESI mass spectrometry in positive ion mode. Internal standard (IS) tetramethylsilane and deuterated dimethyl sulfoxide were utilized in NMR experiments. The Heteronuclear Multiple Quantum Correlation and Heteronuclear Single Quantum Correlation (HMQC and HSQC) spectra of ¹H heteronuclear were acquired using the Bruker AVIII 400 MHz NMR spectrometer to categorize compounds into related groups.

2.5 Selection of wavelength

The intermediates I and others weighing 100 mg were dissolved in methanol and then diluted to a concentration of 10–20 μg/mL. Next, the scanning was performed at a wavelength ranging from 200 to 400 nm.

2.6 Specificity

One of the most important characteristics of HPLC is its specificity, which refers to the analytical method’s capacity to distinguish between the analyte and the other components in the complex mixture [23]. Each compound was dissolved in methanol at a 0.5 mg/mL concentration. Then, 10 μL of each compound was analyzed using HPLC, and the chromatogram was recorded.

2.7 Resolution

Initially, a stock solution of intermediate I was produced in methanol at 1 mg/mL. This solution was intended for use as a reserve solution of reference substance. The other intermediates’ stock methanolic solution (0.2 mg/mL) was prepared. After combining the reference and test stock solutions, methanol was added to obtain a diluted solution containing approximately 0.5 mg/mL of intermediate I and approximately 2.5 μg/mL of other intermediates. The underlined solution was a resolution test solution (system adaptability). Detection was then performed on 10 μL of the solution.

2.8 Precision

Methanolic solution (0.5 mg/mL) of intermediate I was prepared to use as a reference solution. The chromatogram was then observed after 10 μL of each compound was analyzed using HPLC. The comparative standard deviation of peak area normalized content was calculated.

2.9 Linearity

The stock solution of intermediate I was diluted in methanol to concentrations of about 0.04, 0.2072, 0.414, 0.830, 4.144, 20.72, 103.60, 518, and 708 μg/mL as the linear determination solution of intermediate. Then, 10 μL of each solution in the underlined series was analyzed using HPLC, and the chromatogram was recorded. The peak region was plotted against the concentration. The regression equation and correlation coefficient were calculated using the least squares method.

2.10 Quantification and detection limit

The sample stock solutions were serially diluted, and then, 10 μL of each diluted solution was analyzed on HPLC, and the chromatogram was recorded, with the signal-to-noise ratio of about 10:1 as the quantitative limit and the detection limit of about 3:1. The detection and quantification limits of intermediate I was evaluated.

2.11 Stability test

The stock methanolic solution of intermediate I was diluted. Then, 10 μL of each component was examined on HPLC at 0, 2, 4, 6, 8, 10, and 12 h, and the results were recorded in the chromatogram. The RSD of peak area was calculated, and the stability of intermediate I was determined.

2.12 Durability

The adaptive solution of the system was determined at flow rates of 0.9 and 1.1 mL/min and column temperatures of 28 and 32°C. Subsequently, an analysis was conducted to examine the separation of each intermediate peak.

3.1 Method validation parameters

Validation tests were conducted to illustrate the method validation parameters, including wavelength selection, specificity, resolution, linearity, precision, and accuracy.

3.2 Wavelength selection

In this study, the absorption at a wavelength of 210 nm was measured and selected as the detecting wavelength.

3.3 Specificity

Based on the specificity findings, the recorded retention times for starting material II, intermediate III, intermediate IV, intermediate V, intermediate VI, intermediate VII, intermediate VIII, intermediate IX, and BXM were 11.090, 17.508, 19.034, 30.638, 16.893, 28.339, 38.278, 25.655, and 30.593 min, respectively.

3.4 Resolution and precision

The separation between intermediate I and other intermediates was satisfactory, with a degree of separation of ≥1.5, which satisfies the requirements. The precision of the intermediate was evaluated, which corresponded to their chromatogram area. The obtained data showed good precision of the intermediate content in this method. The results are listed in detail in Table 3.

3.5 Linearity

The linearity of this method was evaluated using regression analysis, which revealed that the intermediates and impurities have excellent linearity within a certain range, as shown in Table 4 and Figure 2.

Figure 2

Linearity of intermediate I by HPLC.

3.6 Limit of detection and quantification

Based on the results, the detection and quantification limits for intermediate I were 0.02 and 0.04 µg/mL, respectively.

3.7 Stability and durability

The intermediate I was relatively stable in methanol solution for 12 h, as shown in Table 5. Furthermore, the durability of the method was analyzed by separating each intermediate peak, which revealed that the separation between each peak is greater than 1.5, with good durability, as depicted in Figure 3.

Figure 3

The separation between peaks of the intermediate 1 and impurities.

HPLC evaluations have demonstrated that the effective separation between chromatographic peaks (the separation degree >1.5) can be detected, and the intermediate and its related chemicals can be accurately quantified. In addition, HPLC is a simple, inexpensive, and user-friendly detection method [24,25,26]. The specificity, linearity, range, precision, durability, and other tests yielded satisfactory results. The emphasized data suggested that HPLC could be utilized for regular assessment and quality control of BXM intermediates and related compounds while meeting R&D and production requirements. Kedar et al. published a similar study that utilized reverse phase-HPLC to synthesize, characterize, and quantify impurities in nifedipine and its commercial forms [4,27,28]. Another HPLC-based study was published to ascertain the concentration of alogliptin benzoate in pharmaceutical dosage forms, and this method was validated following ICH and FDA specifications [29]. Their results demonstrated that the HPLC method is robust enough to reproduce precise and accurate results across a broad range of chromatographic parameters [4,29]. Another study was conducted to evaluate the stability of BXM under different stress situations per the guidelines established by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). The observed effects of acid, base hydrolysis, and oxidative stress conditions were stronger on BXM than photo and heat hydrolysis [30]. In an additional investigation, a bioanalytical LC-MS/MS technique was formulated and verified to measure BXA, the biologically active compound of BXM, in the plasma of individuals without medical conditions. This method employed dolutegravir as an IS and involved the precipitation of plasma proteins using acetonitrile. The method has been demonstrated to exhibit linearity throughout the concentration range of 0.5–200.0 ng/mL. Overall, the authors proposed that this particular approach was successfully employed in examining the pharmacokinetics of BXA in a group of healthy human volunteers, demonstrating acceptable repeatability and ruggedness [31]. Our findings agreed with the literature since HPLC proved reliable for identifying and quantifying impurities in BXM and its intermediates.

3.8 Synthesis of impurities

In the synthesis of intermediate V, 2-bromomethyl-3,4-difluorobenzoic acid methyl ester and thiophenol were used as starting materials. Hence, the mechanism of impurity production can be inferred, as depicted in Figure 4. During the process of etherification, the compounds 2-bromomethyl-3,4-difluorobenzoic acid methyl ester and thiophenol underwent oxidation, resulting in the formation of an impurity, namely 3,4-difluoro-2-benzenesulfinyl methyl benzoate. Simultaneously, due to the presence of a small quantity of benzoic acid in the initial substance, the reaction between 2-bromomethyl-3,4-difluorobenzoic acid and benzoic acid resulted in the formation of an impurity known as 2-methyl-3,4-difluoro-2-(benzoyl oxymethyl) benzoate.

Figure 4

Mechanism of impurities formation.

3.9 Characterization of the impurities

A desirable separation effect can be attained by implementing gradient elution. Figure 3 shows that the peak corresponding to intermediate product A1 (t′ = 3.88) is significantly far from the impurity peak (t′ = 6.82). The yellowish impurities were extracted with dichloromethane and then dried under reduced pressure. The characterization of impurities was then performed.

3.9.1 Characterization of impurity 1

MS and NMR measurements were utilized to characterize impurities. Based on the high-resolution mass spectrometric (HRMS) analysis, determining impurity 1 displayed the protonated molecule [M + H]⁺ peak at M/z 311.0544 with an estimated molecular weight of 310.05.

¹H NMR (400 MHz, CDCl₃; 298 K): δ = 7.34(m, 2H), 7.25(m, overlap, 2H), 7.25 (m, overlap, 1H), 7.69(m, 1H), 7.06(m, 1H), 3.83(s, 3H), 4.56(d, J = 2.0 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃; 298 K): δ = 166.01, 151.67, 148.03, 134.72, 132.55, 130.92, 128.83, 127.59, 127.28, 126.21, 115.22, 52.41, 29.90.

The NMR and mass spectrometry results (Figures S1 and S2 of the supplemental data) showed 12 hydrogen atoms and 15 carbon atoms in this molecule. The mass-to-charge ratio (m/z) value for the [M + H]⁺ peak of the ionized product was determined to be 311.0544. The compound is assumed to be 2-((benzoyl oxy) methyl)-3,4-difluorobenzoate (Figure 5) based on a comprehensive analysis.

Figure 5

2-((Benzoyloxy) methyl)-3,4-difluorobenzoic acid methyl ester.

In order to thoroughly examine the resultant impurity structure in BXM, the hydrogen and carbon atoms were subjected to thorough characterization using analytical techniques such as Dept135, ¹⁹F, COSY, HSQC, and HMBC (Table 6, Figures S6–S10). The structure was determined to be 3,4-difluoro-2-benzenesulfinyl methyl benzoate.

Table 6

HSQC and HMBC NMR spectral data of the impurity

Atom	13C	1H
7	166.02	—
	166.01
2	153.47	—
	153.37
	151.77
	151.67
3	149.76
	149.67
	148.11
	148.03
10	134.72	—
11,15	132.55	7.34 (m, 2H)
4	131.00	—
	130.92
12,14	128.83	7.25 (m, overlap, 2H)
13	127.59	7.25 (m, overlap, 1H)
6	127.36	7.69(m, 1H)
	127.33
	127.31
	127.28
5	126.22	—
	126.21
1	115.34	7.06(m, 1H)
	115.22
8	52.41	3.83(s, 3H)
9	29.93	4.56(d, J = 2.0 Hz, 2H)
	29.91
	29.90

Upon comparing the COSY spectrum with the HMQC and DEPT spectra, it was observed that the H (δ 7.34) appeared as a multiplet, and it was proven to be associated with two protons, namely H-11 and H-15, which were associated with the C (δ 132.55). The presence of a multiplet at δ 7.25 was detected, suggesting the existence of two protons (H-12 and H-14) associated with C (δ 128.83). Another multiplet at δ 7.25 was observed, indicating the presence of a H-13 associated with carbon at δ 127.59. The multiplet observed at δ 7.69 correlated with the C at δ 127.28, assigned as H-6. H-1 was observed to appear as a multiplet at (δ 7.06) and to be related to C (δ 115.22). Three protons were detected as a singlet at (δ 3.83), and their relationship to C (δ 52.41) was verified as H-8. The occurrence of two protons at H (δ 4.56) appearing as a doublet, which is correlated with C (δ 29.90), is particularly identified as H-9.

The DEPT spectrum shows a group of primary carbon peaks; the primary carbon peak (δ 52.41) is associated with H-8 (δ 3.83), confirming it as methyl C-8. The spectrum of DEPT exhibits the presence of a group of secondary carbon peaks. The δ 29.90 signal of secondary carbon is attributed to H-9 (δ 4.56) and further confirmed as methylene C-9. Five groups of tertiary carbon peaks could be detected in the spectrum of DEPT. Tertiary carbon peak (δ 115.22) is assigned to H-1 (δ 7.06), confirming it as C-1; tertiary carbon peak (δ 127.28) is related to H-1 (δ 7.69), confirming it as C-6; tertiary carbon peak (δ 127.59) associated with H-1 (δ 7.25), confirming it as C-13; tertiary carbon peak (δ 128.83) associated with H (δ 7.25), confirming it as C-12 and C-14. The tertiary carbon peak (δ 132.55) is associated with H-1 (δ 7.34), confirming it as C-11 and C-15.

3.9.2 Characterization of impurity 2

MS and NMR measurements were utilized to characterize impurities. As shown in Figure S3 of the supplementary data, the HRMS analysis of the detected impurities reveals the protonated molecule [M + H]⁺ peak at M/z 307.0737 with an estimated molecular weight of 306.07.

¹H NMR (400 MHz, CDCl₃; 298 K): δ = 7.54 (tt, J = 7.4, 1.3 Hz, 1H), 7.99 (m,2H), 5.76(d, J = 5.8 Hz, 2H), 7.41 (m, 2H), 7.76(m, overlap, 1H), 7.76(m, 1H), 3.86(s, 3H). ¹³C NMR (100 MHz, CDCl₃; 298 K): δ = 166.10, 165.98, 151.84, 148.93, 133.10, 129.74, 129.69, 128.39, 127.82, 127.12, 126.60, 117.07, 57.03, 52.63.

Combined with NMR (Figures S4 and S5 of the supplementary data) and mass spectrometry analysis, this compound contains 12 hydrogens and 16 carbons. The ionized product’s mass-to-charge ratio of [M + H]⁺ peak is 307.0737. Based on comprehensive analysis, it is speculated that the structural formula of this compound is 3,4-difluoro-2-(benzoyl oxymethyl) methyl benzoate (Figure 6).

Figure 6

3,4-Difluoro-2-(benzoyl oxymethyl) methyl benzoate.

To meticulously verify the resulted structure of impurity present in BXM, hydrogen, and carbon were characterized by Dept135, ¹⁹F, COSY, HSQC, and HMBC (Table 7, Figures S11–S15). The structure was then confirmed as methyl 3,4-difluoro-2-(benzoyl oxymethyl) benzoate.

Table 7

HSQC and HMBC NMR spectral data of the impurity 2

Atom	13C	1H
10	166.10	—
7	166.00	—
	165.98
2	153.63	—
	153.54
	151.93
	151.84
3	150.68	—
	150.60
	149.02
	148.93
14	133.10	7.54 (tt, J = 7.4, 1.3 Hz, 1H)
11	129.74	—
12	129.69	7.99 (m, 2H)
13	128.39	7.41 (m, 2H)
4	127.84	—
	127.82
6	127.20	7.76 (m, 1H)
	127.17
	127.15
	127.12
5	126.68	—
	126.60
1	117.19	7.76 (m, overlap, 1H)
	117.07
9	57.07	5.76 (d, J = 5.8 Hz, 2H)
	57.06
	57.04
	57.03
8	52.63	3.86 (s, 3H)

Upon comparing the COSY spectrum with the HMQC and DEPT spectra, it was observed that the signal corresponding to H-14 at δ 7.54 appeared as a triplet of triplets. This signal was connected with the carbon atom at δ 133.10. The multiplet observed at H (δ 7.99) and the associated proton at C (δ 129.69) can be assigned as H-12 and H-16, respectively. Another multiplet at δ 7.41 for two protons, identified as H-13 and H-15, is related to C (δ 128.39). The proton (H-6) exhibited a multiplet signal at δ 7.76, correlated with the C at δ 127.12. A multiplet corresponding to the proton (H-1) was detected at δ 7.76, which is shown to be correlated with the carbon signal at δ 117.07. Three protons at position H-8 were related to C (δ 52.63) as a singlet at δ 3.86. The presence of two protons of H-9 was detected as a doublet at δ 5.76, which is correlated to the carbon signal at δ 57.03.

The DEPT spectra illustrate the existence of a group of primary carbon peaks. The peak value of primary carbon (δ 52.63) is related to H-8 (δ 3.86), confirming it as methyl C-8. A group of secondary carbon peaks could be detected in the spectrum of DEPT. Furthermore, it has been shown that the methylene group at position C-9 is associated with the secondary carbon peak at δ 57.03, as evidenced by the proton signal at H-9 with a chemical shift of δ 5.76. There are five distinct clusters of tertiary carbon peaks in the DEPT spectrum. The tertiary carbon peak (δ 117.07) is linked to H-1 (δ 7.76), which has been verified as C-1; the tertiary carbon peak (δ 127.12) is linked to H-1 (δ 7.76), verified as C-6; tertiary carbon peak (δ 127.28) are linked to H-1 (δ 7.69), which has also been confirmed as C-6; the tertiary carbon peak (δ 133.10) is associated with H (δ 7.54), which is confirmed as C-14; tertiary carbon peak (δ 129.69) is related to H-1 (δ 7.99), which is identified as C-12 and C-16. The tertiary carbon peak (δ 128.39) is related to H-1 (δ 7.41), confirmed as C-13 and C-15.