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. 2026 Jan 26;40(3):e70346. doi: 10.1002/bmc.70346

Separation and Identification of Impurity of Chlortetracycline Hydrochloride Not Included in the European Pharmacopoeia

Xi Chen 1,✉, Jun Meng 1
PMCID: PMC12835679  PMID: 41588881

ABSTRACT

Drug impurities can affect the clinical efficacy of medicines and may even present substantial health risks. Consequently, the regulation and investigation of such impurities are critically important. In this study, we observed that the chemical structure of impurity E in chlortetracycline hydrochloride, as described in the European Pharmacopoeia, was inconsistent with that of impurity E in the chlortetracycline for system suitability CRS (hereinafter referred to as Impurity 1), as provided by the European Directorate for the Quality of Medicines & HealthCare. To separate and identify the chemical structure of Impurity 1, which is not documented in the European Pharmacopoeia for chlortetracycline hydrochloride, 2D‐LC‐HRMS/MS and HPLC were employed to compare the discrepancies in the chromatographic behavior of Impurity 1 and impurity E, with their chemical compositions further confirmed by NMR and high‐resolution mass spectrometry. Results showed that Impurity 1 and impurity E exhibited inconsistent chemical structures and chromatographic behaviors. Therefore, Impurity 1 and impurity E were identified as two distinct compounds, with impurity E representing the process impurity and Impurity 1 serving as the degradation impurity. These findings provide a comprehensive understanding of impurity in chlortetracycline hydrochloride, as this impurity is reported for the first time.

Keywords: 2D‐LC‐HRMS/MS, chlortetracycline hydrochloride, drug impurity, European pharmacopoeia

1. Introduction

Tetracycline antibiotics (TCs) are natural broad‐spectrum antibiotics derived from Streptomyces strains (Connell et al. 2002; Eric et al. 2021; Yifei and Qian 2019), exerting their bactericidal action through the specific binding to the bacterial 30S ribosomal subunits, thereby inhibiting the growth of peptide chains and disrupting biosynthesis of bacterial proteins (Brodersen et al. 2000; Weiliang et al. 2022). Chlortetracycline, a member of the TCs (Zhao et al. 2020), was first isolated from Streptomyces aureofaciens in 1948 (Duggar 2011), with its specific chemical structure being established in 1953 (Figure 1) (Fabian et al. 2014). Chlortetracycline hydrochloride, a yellow powder, is the hydrochloride salt form of chlortetracycline and represents its most commonly used medical form of chlortetracycline (Suwalsky et al. 1991). Chlortetracycline hydrochloride is most widely utilized as a topical antibiotic in clinical settings for the treatment of skin infections secondary to burns and abrasions (Lowbury and Cason 1954). Additionally, chlortetracycline hydrochloride is frequently employed as an adjunctive treatment for bacterial conjunctivitis and keratitis, typically formulated as an ophthalmic ointment (Kupferman and Leibowitz 1977).

FIGURE 1.

FIGURE 1

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Chemical structure of chlortetracycline.

Drug impurities are defined as substances present in medicines that lack therapeutic activity, affect drug stability or efficacy, or may even pose a threat to human health (Khandavilli et al. 2020). The European Pharmacopoeia (Ph. Eur.) serves as a globally recognized scientific benchmark for the quality control of medicines (Bouin and Wierer 2014). To ensure medicinal products meet the highest standards of quality and safety, the Ph. Eur. attaches great importance to the regulation and control of impurities in medicines and specifies in detail the types, content limits, and detection methods for various impurities. Within the Ph. Eur., the impurities in chlortetracycline hydrochloride are categorized as specified impurities A, B, D, E, G, H, J, K, L, and other detectable impurities C, F, I. It also specifies the liquid chromatography method and corresponding retention time for the identification of the aforementioned specified impurities. However, during a supplementary application for chlortetracycline hydrochloride ophthalmic ointment in accordance with the Ph. Eur., we observed that the chemical structure of impurity E in chlortetracycline hydrochloride, as detailed in the Ph. Eur., was inconsistent with that of impurity E in the chlortetracycline for system suitability CRS (hereinafter referred to as Impurity 1) provided by the European Directorate for the Quality of Medicines & HealthCare (EDQM).

Given the challenging conditions of the chlortetracycline for system suitability CRS in liquid phase separation, 2D‐LC‐HRMS/MS was employed in this study for the qualitative analysis of unknown impurities to avoid the influence of the phosphates in the mobile phase of the mass spectrum system (Liu et al. 2019; Long et al. 2019; Wang et al. 2018). 2D‐LC‐HRMS/MS enables the use of mass spectrometry (MS)‐incompatible mobile phases in the first dimension. Following determination of the target impurity's peak time, the heart‐cutting technique is used to trap and desalt the impurity. The trapped target impurity, stored in the quantitative loop, is then loaded into the second dimension using the mass spectrum‐compatible mobile phase, where it is separated and subsequently detected by high‐resolution MS (Caño‐Carrillo et al. 2023). This method enables the coupling of liquid chromatography with MS to analyze the target impurities without altering the original mobile phase conditions, enables accurate prediction of the impurity structure, while allowing precise localization of impurities under the Ph. Eur. conditions. Consequently, 2D‐LC‐HRMS/MS demonstrates superior selectivity and separation capacity compared to conventional LC–MS, rendering it a valuable tool for drug impurity profiling (Wang et al. 2017). This study aims to, for the first time, isolate and determine the impurity structure which is not documented in Ph. Eur.

2. Materials and Methods

2.1. Chemicals

MS grade methanol, perchloric acid, and formic acid were purchased from Sigma Inc. (USA). MS grade acetonitrile was from Anaqua Global International Inc., Limited (Cleveland, USA). Purified water was supplied by China Resources C'estbon Beverage (China) Co. Ltd. The raw material of chlortetracycline hydrochloride was provided by Fukang Pharmaceutical Co. Ltd. (Fujian, China). The chlortetracycline for system suitability CRS was obtained from the EDQM. Deuterated dimethyl sulfoxide (DMSO‐d6) was purchased from Shanghai Haohong Bio‐pharmaceutical Technology Co. Ltd. The HPLC grade formic acid and acetonitrile were purchased from Concord Technology (Tianjin) Co. Ltd.

2.2. Instrumentation and Software

NMR spectra were taken on a Bruker Avance III 500 MHz NMR spectrometer (Bruker, Germany). MS data were recorded using the Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, USA). For HPLC analysis, the UltiMate 3000 UHPLC DGLC consisting of the UltiMate 3000 pump, UltiMate 3000 autosampler, and UltiMate 3000 diode array detector were employed (Thermo Fisher Scientific, USA). Data analysis was performed using the Thermo Xcalibur Qual Browser 4.0.

2.3. 2D‐LC‐HRMS/MS Analysis

2.3.1. Sample Preparation

The chlortetracycline for system suitability CRS was configured as a 2 mg/mL solution with the initial mobile phase for 2D‐LC‐HRMS/MS system analysis.

2.3.2. The First‐Dimensional Chromatographic Condition

Chromatographic separation was performed on an Acclaim C18 chromatographic column (4.6 × 150 mm, 5 μm; Thermo Fisher Scientific) with a detection wavelength of 280 nm. The column temperature was set to 45°C, and the injection volume was 10 μL. The mobile phase A was water‐perchloric acid‐dimethyl sulfoxide (72.5: 5: 22.5, v/v/v) and the mobile phase B was water‐perchloric acid‐dimethyl sulfoxide (25: 5: 70, v/v/v). The following gradient with a flow rate of 0.8 mL/min was run: the mobile phase B was increased from 0% to 100% for 36 min, followed by a decrease in mobile phase B from 100% to 0% within 4 min. Then, the system was re‐equilibrated with 0% B for 2 min.

2.3.3. The 2D Desalt Chromatography Condition

The analytes were chromatographically separated using an Ultimate AQ‐C18 column (3.0 × 50 mm, 5 μm; Thermo Fisher Scientific) operating at 30°C. Gradient elution procedure was performed for the separation with acetonitrile (A) and 0.1% formic acid in water (B) delivered at a flow rate of 0.3 mL/min. The optimized elution program was as follows: 0–18 min, 99% B; 18–36 min, 99%–20% B; 36–46 min, 20% B; 46–56 min, 20%–99% B; 70 min, 99% B.

2.3.4. MS Parameters

The Q Exactive Focus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was operated in ESI‐positive mode for detection. The parameters were as follows: ion spray voltage, 3.8 kV; metal capillary temperature, 320°C; spray temperature, 280°C; sheath gas pressure, 30 arb; aux gas pressure, 10 arb; S‐lens RF level, 55; normalized collision energy (NCE), 30. The data were collected and processed using Thermo Xcalibur Qual Browser 4.0.

2.4. HPLC Analysis

2.4.1. Sample Preparation

The chlortetracycline for system suitability CRS was configured into a 2 mg/mL solution with the initial mobile phase to obtain the chlortetracycline solution for system suitability CRS. The preparation of demeclocycline reaction positioning solution involved dissolving 100 mg of demeclocycline hydrochloride in 10 mL of hydrochloric acid solution (0.01 M) and letting the mixture stand for 6 h at room temperature. Then, the appropriate amount of the above mixture was taken and diluted with the initial mobile phase. Demeclocycline was used to prepare impurity E, which was subsequently dissolved using the initial mobile phase to produce its positioning solution. The initial mobile phase dissolved Impurity 1 and chlortetracycline raw material, yielding the corresponding positioning solution.

2.4.2. Chromatographic Conditions

The chromatographic separation was performed on an Acclaim C18 column (150 × 4.6 mm, 5 μm particles, Thermo Fisher Scientific) with a detection wavelength of 280 nm, using a gradient solvent system for water, perchloric acid and dimethyl sulfoxide. Mobile phase A was water‐perchloric acid‐dimethyl sulfoxide (72.5: 5: 22.5, v/v/v), B was water‐perchloric acid‐dimethyl sulfoxide (25: 5: 70, v/v/v). The separation was carried out at 45°C with an injection volume of 5 μL, and the mobile phase flow rate was 0.8 mL/min. The following gradient elution was used: 0%–100% of B at 0–36 min, 100% of B at 36–62 min, 100%–0% of B at 62–65 min, 0% of B at 65–70 min.

3. Results and Discussion

3.1. 2D‐LC‐HRMS/MS Analysis

Two‐dimensional liquid chromatography has been demonstrated to have better selectivity, peak capacity, and resolution than one‐dimensional liquid chromatography and is widely used for the detection of complex samples (Montero and Herrero 2019; Zhou et al. 2020). During the experiment carried out using 2D‐LC‐HRMS/MS, the retention time of Impurity 1 was determined by the first‐dimensional chromatographic positioning to be 16.81 min. In the next second‐dimensional desalting analysis, the valve switching time in the liquid chromatographic method was set to 16.71–16.95 min for heart‐cutting Impurity 1. Impurity 1 was then cut into the mass spectrum for 18 min. The primary and secondary mass spectra of Impurity 1 were shown in Figure 2A,B, respectively.

FIGURE 2.

FIGURE 2

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The MS1 (A) spectrum and MS2 spectrum (B) in the positive ionization of Impurity 1.

As demonstrated in Table 1, Figure 2, the first‐dimensional quasi‐molecular ion peak of the Impurity 1 was found according to the retention time in the extracted ion chromatogram (EIC) of m/z 444.16444, which was consistent with its molecular structure. The secondary parent ion was 444.1642 and the secondary fragmentation ions were 170.08096 and 257.08038, respectively. The protonated molecule of Impurity 1 at m/z 444.16529 fragmented into the product ion at m/z 257.08084 with a loss of 187 Da and fragmented into the product ion at m/z 170.08117 at a loss of 274 Da. Therefore, plausible structure, as shown in Figure 3, was proposed based on the fragmentation pattern obtained from accurate MS2 spectra.

TABLE 1.

Analysis results of 2D‐LC‐HRMS/MS.

First‐dimensional quasi‐molecular ion peak (m/z) Secondary parent ion (m/z) Secondary fragmentation ions (m/z)
Impurity 1 444.16444 444.1642 170.08096, 257.08038
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FIGURE 3.

FIGURE 3

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Proposed fragmentation pathways of Impurity 1 by MS2.

3.2. Structural Identification of Impurity 1

Impurity 1 was a degradation impurity produced during storage of chlortetracycline hydrochloride. Its molecular formula was assigned as C23H25NO8 which was determined by its ESI‐MS at m/z 444.16437 [M + H]+ (calculated for 444.16584). 1H NMR spectra displayed characteristic three adjacent protons on the benzene ring at 7.084–7.099, 1H, d, J = 7.5 Hz; 7.503–7.535, 1H, dd, J = 7.5, 7.5 Hz; 6.895–6.910, 1H, d, J = 7.5 Hz indicates the absence of chlorine substituent on the benzene ring. It is confirmed that there is no chlorine isotopic ion in MS. 1H NMR spectra displayed a methyl singlet at δH 2.137 and the 13C NMR spectra exhibited carbon resonances at δC 31.59 while carbon signal at 190 indicating the existence of acetyl group. The 1H and 13C NMR spectra of Impurity 1 (Table 2), the structure of Impurity 1 was presented in Figure 4A it was in agreement with the (4S,4aS,5aS,6S,12aS)‐2‐acetyl‐4‐(dimethylamino)‐3,6,10,12,12a‐pentahydroxy‐6‐methyl‐4a,5a,6,12a‐tetrahydrotetracene‐1,11(4H,5H)‐dione.

TABLE 2.

1H and 13C NMR data of Impurity 1 and impurity E (DMSO‐d6).

Position Impurity 1 Impurity E
δ H (ppm) δ C (ppm) δ H (ppm) δ C (ppm)
1 190.64, C 191.57, C
2 106.83, C 97.97, C
2‐CO— 193.36, C 160.68, C
2‐COCH3 2.137, 3H, s 31.59, CH3
3 183.13, C 179.14, C
3‐OH 11.983, 1H, s 9.086, 1H, s
4 3.296, 1H, m 68.43, CH 4.030–4.037, 1H, J = 3.5 Hz 65.05, CH
4‐N (CH3)2 2.721, 3H x 2, s 42.35, CH3 2.616, 3H, s; 2.572, 3H, s 44.65, CH3
4a 2.426–2.436, 1H, m 32.79, CH 2.630–2.652, 1H, m 40.38, CH
5 1.835–1.865; 1.989–2.026, 2H, m 23.16, CH2 1.727–1.778; 1.803–2.001, 2H, m 22.85, CH2
5a 2.836–2.868, 1H, m 40.24, CH 2.839–2.876, 1H, m 37.82, CH
6 76.29, C 5.294–5.306, 1H, J = 6.0 Hz 64.66, CH
6‐CH3 1.523, 3H, s 28.52, CH3
6a‐OH 3.705, 1H, s 4.173–4.696, 1H, m
6a 148.51, C 137.04, C
7 7.084–7.099, 1H, d, J = 7.5 Hz 115.11, CH 122.60, C
8 7.503–7.535, 1H, dd, J = 7.5, 7.5 Hz 136.58, CH 7.576–7.594, 1H, J = 9.0 Hz 136.27, CH
9 6.895–6.910, 1H, d, J = 7.5 Hz 115.52, CH 6.938–60,956, 1H, J = 9.0 Hz 119.25, CH
10 161.84, C 141.30, C
10‐OH 15.246, 1H, s 11.985, 1H, s
10a 117.29, C 116.66, C
11 192.69, C 192.84, C
11a 110.28, C 104.90, C
12 181.31, C 8.594, 1H, s 173.42, C
12‐OH 9.518, 1H, s 11.985, 1H, s
12a 76.29, C 76.23, C
12a‐OH 4.853, 1H, s 6.724, 1H, s
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FIGURE 4.

FIGURE 4

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Chemical structure of Impurity 1 (A) and impurity E (B).

3.3. Structural Identification of Impurity E

Impurity E was a process impurity produced during the synthesis of chlortetracycline hydrochloride. The chemical structure analysis showed that impurity E in the Ph. Eur. was an isomer of demeclocycline (Figure 4B). Consequently, impurity E was prepared by chemical isolation from raw medicine of demeclocycline as shown in Figure 5. The molecular formula of the home‐made impurity E was determined as C21H21ClN2O8 based on the ESI‐MS at m/z 465.10577 [M + H]+ (calculated for 465.10647). Analysis of the 1H and 13C NMR spectra of the home‐made impurity E (Table 2), 1H NMR and 13C NMR spectra data are basically consistent with those of chlortetracycline, except for an additional proton signal at δH 5.294–5.306, 1H, J = 6.0 Hz, corresponding to carbon signal at δC 64.66 while a methyl signal at 6 position of chlortetracycline was not observed. This indicates that it is an isomer of demeclocycline. it was consistent with the impurity E [(4R,4aS,5aS,6S,12aS)‐7‐chloro‐4‐(dimethylamino)‐3,6,10,12,12a‐pentahydroxy‐1,11‐dioxo‐1,4,4a,5,5a,6,11,12a‐octahydrotetracene‐2‐carboxamide(4‐epidemethylchlortetracycline)] of chlortetracycline hydrochloride in the Ph. Eur.

FIGURE 5.

FIGURE 5

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Preparation route of impurity E.

3.4. HPLC Analysis

Analyze the chromatographic separation of (a) Impurity 1 positioning solution, (b) demeclocycline reaction positioning solution, (c) the chlortetracycline solution for system suitability CRS, (d) impurity E positioning solution and (e) chlortetracycline raw material positioning solution in the chromatographic system as detailed in Figure 6. In the demeclocycline reaction positioning solution, two peaks were eluted with a retention time of about 17.65 min for demeclocycline and about 13.62 min for impurity E prepared by separation. The impurity E obtained from the separation and preparation was determined by 1H‐NMR, 13C‐NMR and MS to be consistent with the chemical structure of impurity E as specified by the European Pharmacopoeia. 2D‐LC‐HRMS/MS method was used to confirm the structure of Impurity 1 (tR = 15.15 min) provided by system suitability CRS. By comparing a, c, and e, it was found that the chromatographic behavior of the chlortetracycline raw material positioning solution was consistent with that of the chlortetracycline solution for system suitability CRS. Besides, there was an impurity chromatographic peak at the retention time of about 15.15 min, which indicated that Impurity 1 rather than impurity E existed in the chlortetracycline raw material.

FIGURE 6.

FIGURE 6

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The HPLC chromatographic spectrum of (a) Impurity 1 positioning solution, (b) demeclocycline reaction positioning solution, (c) the chlortetracycline solution for system suitability CRS, (d) impurity E positioning solution and (e) chlortetracycline raw material positioning solution.

3.5. Limitations

There are innate limitations in our study. Firstly, we did not perform quantitative analysis of Impurity 1. In the future, we plan to obtain a high‐purity standard of Impurity 1 through the preparation of two‐dimensional liquid chromatography separation. Based on this standard, we aim to establish a quantitative detection method to systematically investigate the relative content, concentration range, and stability of Impurity 1 in samples of chlortetracycline hydrochloride from different sources (clinical, commercial) and batches. Secondly, for qualitative targets, the 70 min two‐dimensional run time on a 5 cm chromatography column is relatively long. We will further assess the feasibility of shortening the two‐dimensional gradient and validate in future studies whether the shortened gradient can still achieve the same level of qualitative accuracy and separation efficiency for the target components, thereby enhancing the method's practicality. Thirdly, due to the depletion of the standard sample of impurity E and Impurity 1, we did not perform a brief 1H NMR overlay experiment. This supplementary NMR experiment will be conducted in future studies.

4. Conclusions

To the best of our knowledge, this is the first study to report on Impurity 1 in chlortetracycline, presenting qualitative analyses using 2D‐LC‐HRMS/MS for system suitability CRS. These results demonstrate that the molecular weight of Impurity 1 was 443, which was not in conformity with the impurity E specified in the Ph. Eur. According to the Ph. Eur., the chemical structure of impurity E suggests that it is formed via the degradation of demeclocycline under acidic conditions. The chemical structures of Impurity 1 and impurity E, following purification, were confirmed by nuclear magnetic resonance‐hydrogen spectrum, nuclear magnetic resonance‐carbon spectrum, and MS. HPLC analyses of Impurity 1, impurity E, and the chlortetracycline solution for system suitability CRS further substantiated that the chemical name of Impurity 1 in the chlortetracycline for system suitability CRS, as provided by the EDQM, is (4S,4aS,5aS,6S,12aS)‐2‐acetyl‐4‐(dimethylamino)‐3,6,10,12,12a‐pentahydroxy‐6‐methyl‐4a,5a,6,12a‐tetrahydrotetracene‐1,11(4H,5H)‐dione. This was different from impurity E of chlortetracycline hydrochloride in the Ph. Eur. whose chemical name was (4R,4aS,5aS,6S,12aS)‐7‐chloro‐4‐(dimethylamino)‐3,6,10,1212a‐pentahydroxy‐1, 11‐dioxo‐1,4,4a,5,5a,6,11,12a‐octahydrotetracene‐2‐carboxamide(4‐epidemethylchlortetracycline). Our study provides a comprehensive understanding of Impurity 1 which is not listed as an impurity of chlortetracycline hydrochloride in the Ph. Eur. It demonstrates the significance of developing analytical methods and establishing quality standards. Whereas the accuracy of the chemical structure of impurity E in the Ph. Eur., as well as the potential issue with Impurity 1 in the chlortetracycline for system suitability CRS provided by the EDQM, necessitates further investigation and feedback to the EDQM.

Author Contributions

Xi Chen: conceptualization, writing – review and editing, methodology. Jun Meng: methodology, investigation, writing – original draft, resources.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Chromatogram of Impurity 1 in 2D‐LC‐HRMS/MS.

Figure S2: Chromatogram of Impurity 1 positioning solution.

Figure S3: Chromatogram of demeclocycline reaction positioning solution.

Figure S4: Chromatogram of the chlortetracycline solution for system suitability CRS.

Figure S5: Chromatogram of impurity E positioning solution.

Figure S6: Chromatogram of chlorotetracycline raw material positioning solution.

Figure S7: bmc70346‐sup‐0001‐Supporting_Information .docx. 1H NMR spectrum of Impurity 1 (DMSO‐d6, 500 MHz).

Figure S8: bmc70346‐sup‐0001‐Supporting_Information .docx. 13C NMR spectrum of Impurity 1 (DMSO‐d6, 500 MHz).

Figure S9: HRESI (+) MS spectrum of Impurity 1.

Figure S10: bmc70346‐sup‐0001‐Supporting_Information .docx. 1H NMR spectrum of impurity E (DMSO‐d6, 500 MHz).

Figure S11: bmc70346‐sup‐0001‐Supporting_Information .docx. 13C NMR spectrum of impurity E (DMSO‐d6, 500 MHz).

Figure S12: HRESI (+) MS spectrum of impurity E.

BMC-40-e70346-s001.docx (2MB, docx)

Acknowledgments

We sincerely thank the “Baiyunshan Hejigong Pharmaceutical Factory, Guangzhou Baiyunshan Pharmaceutical Holdings Co. Ltd”.

Chen, X. , and Meng J.. 2026. “Separation and Identification of Impurity of Chlortetracycline Hydrochloride Not Included in the European Pharmacopoeia.” Biomedical Chromatography 40, no. 3: e70346. 10.1002/bmc.70346.

Data Availability Statement

All data included in this study are available upon request by contact with the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: Chromatogram of Impurity 1 in 2D‐LC‐HRMS/MS.

Figure S2: Chromatogram of Impurity 1 positioning solution.

Figure S3: Chromatogram of demeclocycline reaction positioning solution.

Figure S4: Chromatogram of the chlortetracycline solution for system suitability CRS.

Figure S5: Chromatogram of impurity E positioning solution.

Figure S6: Chromatogram of chlorotetracycline raw material positioning solution.

Figure S7: bmc70346‐sup‐0001‐Supporting_Information .docx. 1H NMR spectrum of Impurity 1 (DMSO‐d6, 500 MHz).

Figure S8: bmc70346‐sup‐0001‐Supporting_Information .docx. 13C NMR spectrum of Impurity 1 (DMSO‐d6, 500 MHz).

Figure S9: HRESI (+) MS spectrum of Impurity 1.

Figure S10: bmc70346‐sup‐0001‐Supporting_Information .docx. 1H NMR spectrum of impurity E (DMSO‐d6, 500 MHz).

Figure S11: bmc70346‐sup‐0001‐Supporting_Information .docx. 13C NMR spectrum of impurity E (DMSO‐d6, 500 MHz).

Figure S12: HRESI (+) MS spectrum of impurity E.

BMC-40-e70346-s001.docx (2MB, docx)

Data Availability Statement

All data included in this study are available upon request by contact with the corresponding author.

Articles from Biomedical Chromatography are provided here courtesy of Wiley

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