Solvents Influence ¹H NMR Chemical Shifts and Complete ¹H and ¹³C NMR Spectral Assignments for Florfenicol

Abstract

Florfenicol (FFC) is an important and widely used veterinary drug, and its structure has been characterized by nuclear magnetic resonance (NMR) spectroscopy. The study aimed to investigate the influences of solvent type, solvent concentration, and temperature on the chemical shifts of the ¹H NMR of FFC. The results showed that different types of solvents significantly affected the chemical shifts, especially the chemical shifts of 2-H, 3-H, 5-H, and the active protons. When DMSO-d ₆ is used as the solvent, there is no significant difference in the chemical shifts of FFC with a concentration ranging from 20 to 250 mmol/L; however, as the temperature increases, the chemical shifts of the active protons move to a higher field. Besides, the NMR spectroscopic data and structural analysis of FFC were refined by ¹H, ¹³C, distortionless enhancement by polarization transfer-135 (DEPT-135), ¹H–¹H correlation spectroscopy (¹H–¹H COSY), phase-sensitive gradient heteronuclear singular quantum correlation (gHSQC), and heteronuclear multiple bond correlation (gHMBC) NMR spectroscopy using DMSO-d ₆ as a solvent. The study will help with qualitative and quantitative analysis of FFC in the future.

Keywords

florfenicol - nuclear magnetic resonance - solvents - solvent concentrations - chemical shifts

Introduction

Florfenicol (FFC) ([Fig. 1]), a derivative of chloramphenicol, is an important broad-spectrum antibiotic that can diffusely enter into bacterial cells through fat solubility. FFC mainly acts on the 50s subunit of the bacterial 70s ribosome to inhibit peptidyl transferase and subsequent peptide chain elongation by blocking peptidase growth and peptide chain formation, thus, preventing the synthesis of bacterial proteins. FFC is frequently used to treat infectious infections in aquatic, livestock, and poultry, due to its advantages including potent antibacterial action, low risk of drug resistance, good absorption in vivo, high safety, high bioavailability, etc.[1] [2] [3] However, the growing use of FFC may be associated with adverse effects. Hassanin et al found that FFC-treated chickens had a significantly high number of adipocytes in the bone marrow, and the hematopoietic cell lines were moderately atrophied,[4] confirming the influence of FFC in hematopoietic and immune functions. Yun et al demonstrated a negative impact of oral administration of FFC on intestinal mucosal barriers, immune system, and intestinal microbiota.[5] Wang et al suggested a promoting effect of FFC on the oxidative stress response, which plays an important role in inducing kidney injury in broiler chickens.[6]

Fig. 1 Structure of FFC. FFC, florfenicol.

To protect food health and safety, the detection of FFC residue in animal and meat-derived foods has been gradually emphasized.[7] [8] [9] [10] [11] [12] [13] [14] Chloramphenicol analogs usually have low residue levels in aquatic products, thus, it is necessary to use high sensitivity and low detection limit assays to detect them. Li et al calculated the total residue levels of FFCs in eggs using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to quantify the metabolite florfenicolamide.[15] Wang et al established a simple, rapid, and novel ultra-performance liquid chromatography with fluorescence detection (UPLC-FLD) method to determine methylsulfonamides, FFC, and its metabolite florfenicolamide residues in poultry eggs.[16] Patyra and Kwiatek used a liquid chromatography and diode array detector to simultaneously determine FFC and methylsulfonamides in medicinal feed.[17]

Nuclear magnetic resonance (NMR) is characterized by its speed and ease of use. NMR uses the relationship between the number of protons of a specific group in the molecule and the corresponding spectral peaks to quickly, accurately, and quantitatively analyze the components without the aid of the standards. With the advancement of NMR techniques in terms of hardware and software, quantitative analysis using NMR techniques has been paid increasing attention and has been used to study the changes in drugs in solvents.[18] [19] [20] [21] [22] [23] [24] [25] In this study, the NMR technique was used to analyze the spectral data and the structure of FFC. Besides, the effects of solvents, FFC concentration, and temperature on the chemical shifts of the FFC ¹H NMR spectra were assessed. All ¹H and ¹³C NMR chemical shifts were determined by advanced one-dimensional (1D) and two-dimensional (2D) NMR.

Experimental

Materials and Reagents

FFC used for spectral measurement was supplied by Zhejiang Hisoar Pharmaceutical Co. Ltd. (Taizhou, Zhejiang) with a purity of 99%. Chloroform-d (CDCl₃, 99.9 atom% D), acetonitrile-d ₃ (ACN-d ₃, 99.8 atom% D), acetic acid-d₄ (CD₃COOD, 99.5 atom% D), methanol-d ₄ (CD₃OD, 99.8 atom% D), acetone-d ₆ (CD₃COCD₃, 99.9 atom% D), methyl sulfoxide-d ₆ (DMSO-d ₆, 99.9 atom% D), N,N-dimethylformamide-d ₇ (DMF-d ₇, 99.5 atom% D), and pyridine-d ₅ (C₅D₅N, 99.5 atom% D) were purchased from Cambridge Isotope Laboratories Inc.(Cambridge, United States).

Apparatus and Conditions

The advanced 1D and 2D NMR spectra were recorded at 303 K on a Varian Mercury-Plus 400 MHz spectrometer (Varian, United States) operating at frequencies of 400.13 and 100.62 MHz using 5 mm ID probe for ¹H/¹³C, respectively. Tetramethylsilane (δ 0.00 ppm) and DMSO-d ₆ (δ 39.50 ppm) were used as the reference for the measurement of ¹H and ¹³C NMR chemical shifts. Eight solutions in different solvents (CDCl₃, ACN-d ₃, CD₃COOD, CD₃OD, CD₃COCD₃, DMSO-d ₆, DMF-d ₇, C₅D₅N) with FFC concentrations at 50 mmol/L, six solutions (20, 50, 100, 150, 200, 250 mmol/L) in DMSO-d ₆, and three solutions (20, 100, 250 mmol/L) in DMSO-d ₆ at different temperatures (303, 308, 313, 318, 323, 328, 333, 338, 343 K) were prepared for investigating the influences of ¹H NMR chemical shifts of FFC. ¹H NMR spectra were recorded with acquisition times of 2.5 seconds using a 45-degree pulse and ¹³C spectra were obtained with acquisition times of 1.2 seconds using a 30-degree pulse. The distortionless enhancement by polarization transfer (DEPT) spectrum was recorded using relaxation delay times of 2 seconds by using a shaped pulse for a 180-degree pulse on the f1 channel with a 135-degree read pulse to give CH, CH₃ positive, and CH₂ negative. The 2D NMR spectra were recorded with standard pulse sequences supplied with the TopSpin 3.6.5 spectrometer software (Bruker DESKTOP-G4TBOV8). The ¹H–¹H COSY spectrum was recorded using one transient per t ₁ increment with relaxation delay times of 2 seconds, spectral widths of 4587.2 Hz, and acquisition times of 0.223 seconds in t ₂ with 128 complex t ₁ increments. The phase-sensitive gradient heteronuclear singular quantum correlation (gHSQC) spectrum was obtained with polarization transfer and refocusing delays optimized for ¹ J _CH = 150 Hz; four transients were recorded for each of 128 complex t ₁ increments with relaxation delay times of 2 seconds, spectral widths of 4,587.2/17,094.0 Hz, and acquisition times of 0.223 seconds in t ₂. The heteronuclear multiple bond correlation (gHMBC) spectrum was obtained with polarization transfer delays optimized for ¹ J _CH = 150 Hz, ⁿ J _CH = 6 Hz; four transients were recorded for each of 400 complex t ₁ increments with relaxation delay times of 2 seconds, spectral widths of 4,587.2/24,154.6 Hz, and acquisition times of 0.223 seconds in t ₂. All 1D and 2D spectra were processed using Mestrelab's Mnova software, version 7.1.0-9185 (Santiago de Compostela, Spain).

Results and Discussion

The Influences of Solvents on ¹H NMR of FFC

FFC dissolved in different solvents was used to identify changes in chemical shifts in ¹H NMR spectra. Approximately 9 mg of FFC was dissolved in 500 μL different pure solvents forming a concentration of 50 mmol/L, respectively, but the solubility of FFC in the CDCl₃ was very poor and saturated in the solution. Our data showed that the solvent causes a significant change in ¹H NMR chemical shifts ([Fig. 2]). The entire elucidation of the hydrogen protons of the FFC in various solvents is displayed in [Table 1]. When CD₃OD and CD₃COOD were used, the active hydrogen protons disappeared due to the exchange with the deuterium hydrogen which made 5-H exhibit a doublet peak, while in other solvents (ACN-d ₃, CD₃COCD₃, DMSO-d ₆, DMF-d ₇), the 5-H showed a doublet of doublet peak due to the coupling with both 4-H and the O–H. As a result, the O–H proton showed a double peak. The signals of O–H, 4-H, and 3-H were overlapping when using C₅D₅N as a solvent, which led to the difficulty of integration. Moreover, it was strange that the 5-H showed a single peak. The signals of 4-H and 3-H were overlapping when using CD₃COOD as a solvent, which made it difficult to determine the coupling constants. When CDCl₃ was used as a solvent, the signals of 4-H and 3-H were separated clearly. However, the coupling of 5-H with the O–H proton was not observed, which showed a doublet peak and a broad singlet peak, respectively. The N–H proton coupling with the 4-H was obvious. As far as 2-H was concerned, its chemical shift increased with the solvents' order as follows: CDCl₃, ACN-d ₃, CD₃COOD, CD₃OD, CD₃COCD₃, DMSO-d ₆, DMF-d ₇, C₅D₅N, and the chemical shift moved to a low frequency from δ 5.81 to δ 6.81 ppm. All these results substantiate the solvent effect. The solvent effect mainly has the following aspects: firstly, different solvents have different solvent magnetic permeability, so FFCs are subjected to different magnetic field strengths, which affects the δ value. Secondly, the solvent molecules can be close to the solute molecules, which changes the electron cloud density of the solute molecules and produces a deshielding effect. In addition, the magnetic anisotropic effect of solvent molecules leads to shielding and deshielding effects on different parts of the solute molecules. Moreover, solvent molecules form hydrogen bonds with solute molecules, which can also affect the chemical shift of the hydrogen nucleus.[22] [26] In particular, the aromatic solvent, C₅D₅N, showed the most pronounced changes.

Table 1 The ¹H NMR chemical shifts of FFC in different solvents

Solvent/proton number	CDCl3	ACN-d 3	CD3COOD	CD3OD	CD3COCD3	DMSO-d 6	DMF-d 7	C5D5N
2-H	5.81 (s)	6.03 (s)	6.17 (s)	6.20 (s)	6.35 (s)	6.44 (s)	6.59 (s)	6.81 (s)
3a′-H	4.48–4.52 (dd, 4.4, 9.2)	4.29–4.42 (m)	4.43–4.75 (m)	4.35–4.42 (m)	4.39–4.49 (m)	4.22–4.33 (m)	4.39–4.49 (m)	4.96–5.06 (m)
3a′′-H	4.56–4.63 (dd, 6.4, 9.2)	4.49–4.53 (dd, 6.8, 9.6)	4.43–4.75 (m)	4.47–4.51 (dd, 6.8, 9.2)	4.54–5.58 (dd, 6.4, 9.2)	4.41–4.45 (dd, 7.2, 9.2)	4.52–4.56 (dd, 6.8, 8.8)	4.63–4.67 (dd, 6.4, 9.2)
3b′-H	4.56–4.63 (dd, 4.4, 9.6)	4.56–4.59 (dd, 5.6, 9.2)	4.43–4.75 (m)	4.58–4.63 (m)	4.62–4.67 (m)	4.56–4.60 (dd, 5.2, 8.8)	4.66–4.71 (m)	4.74–4.79 (m)
3b′′-H	4.67–4.71 (dd, 6.8, 9.6)	4.67–4.71 (dd, 6.0, 9.6)	4.43–4.75 (m)	4.70–4.74 (dd, 6.0, 9.2)	4.74–4.78 (dd, 6.0, 9.2)	4.68–4.71 (dd, 5.2, 8.8)	4.78–4.82 (dd, 5.6, 9.2)	4.89–4.93 (dd, 6.4, 9.2)
4-H	4.27–4.37 (m)	4.29–4.42 (m)	4.43–4.75 (m)	4.35–4.42 (m)	4.39–4.49 (m)	4.22–4.33 (m)	4.39–4.49 (m)	4.96–5.06 (m)
5-H	5.24 (d, 2.8)	5.08 (dd, 4.0, 7.6)	5.22 (d, 3.2)	5.08 (d, 3.2)	5.22 (dd, 4.0, 7.2)	4.98 (dd, 4.0, 7.6)	5.16 (dd, 4.4, 7.6)	5.52 (s)
7-H 11-H	7.58 (d, 8.4)	7.60 (d, 8.4)	7.65 (d, 8.4)	7.65 (d, 8.4)	7.70 (d, 8.4)	7.60 (d, 8.4)	7.70 (d, 8.0)	7.89 (d, 8.0)
8-H 10-H	7.89 (d, 8.4)	7.85 (d, 8.4)	7.92 (d, 8.4)	7.88 (d, 8.4)	7.89 (d, 8.4)	7.83 (d, 8.4)	7.90 (d, 8.4)	8.15 (d, 8.4)
12-H	3.04 (s)	3.02 (s)	3.09 (s)	3.07 (s)	3.07 (s)	3.15 (s)	3.19 (s)	3.19 (s)
NH	6.97 (d, 8.4)	7.05 (d, 8.0)	–	–	7.64 (d, 6.8)	8.55 (d, 8.8)	8.51 (d, 8.4)	9.55 (d, 8.0)
OH	2.77 (s)	4.05 (d, 4.8)	–	–	5.29 (d, 4.4)	6.11 (d, 4.4)	6.17 (d, 4.4)	4.86 (br)

Abbreviation: FFC, florfenicol.

Fig. 2 ¹H NMR spectra of FFC at different solvents. (i)–(viii): CDCl₃, ACN-d ₃, CD₃COOD, CD₃OD, CD₃COCD₃, DMSO-d ₆, DMF-d ₇, C₅D₅N, respectively; 400 MHz, temperature 303 K. FFC, florfenicol.

The Influences of Solvent Concentrations on ¹H NMR of FFC

Generally, the concentration will also have a certain effect on the ¹H NMR chemical shift, acting mainly on changes in solution viscosity, changes in ion pairs, or changes in aggregation states.[27] In this work, six different concentrations of FFC solutions in DMSO-d ₆ (20, 50, 100, 150, 200, 250 mmol/L) were used to further investigate the influence of concentrations on the ¹H NMR chemical shifts of FFC; however, no dramatical changes were found ([Fig. 3]).

Fig. 3 ¹H NMR spectra of FFC at different concentrations in DMSO-d ₆ solvent. (i)–(vi): 20, 50, 100, 150, 200, 250 mmol/L respectively; 400 MHz, temperature 303 K. FFC, florfenicol.

The Influences of Temperatures on ¹H NMR of FFC

To evaluate the influences of temperature, three solutions at different concentrations (20, 100, 250 mmol/L) of FFC in DMSO-d ₆ were prepared for further experiments. The results showed that the samples with different concentrations at different temperatures exhibited similar changes in chemical shifts of active protons. The chemical shift of the O–H proton moved to a high frequency from δ 8.56 to δ 8.32 ppm when the temperature rose from 303 to 343 K. Every 5-degree temperature increase led to a 0.03 ppm chemical shift to the high frequency at the same time, the chemical shift of the N–H proton moved to a high frequency from δ 6.12 to δ 5.95 ppm, with 0.02 ppm of the chemical shift moving to the high field as the temperature increased 5 degrees. At high temperatures the thermal motion of the molecule is intensified, the degree of hydrogen bonding is reduced, and the density of the electron cloud around the hydrogen nucleus is increased making the hydrogen protons move into the higher field, and hence the corresponding chemical shift is reduced. The above changes were only found in active protons, while the chemical shifts of other protons did not significantly exhibit such changes. It was confirmed that the influence of temperature on active protons of FFC was greater than other protons ([Fig. 4]).

Fig. 4 ¹H NMR spectra of FFC at different temperatures in DMSO-d ₆ solvent. (i) – (ix): 303, 308, 313, 318, 323, 328, 333, 338, 343, respectively; 400 MHz, 20 mmol/L. FFC, florfenicol.

The Structure Elucidation of FFC by Comprehensive NMR Analysis

The structure of FFC was elucidated with DMSO-d ₆ as a solvent. Assignments of all ¹H NMR chemical shifts are summarized in [Table 2]. Hydrogen and carbon atoms were assigned by using ¹H NMR, full decoupling ¹³C NMR, DEPT-135, ¹H–¹H COSY, gHSQC, and gHMBC. Among 14 hydrogen proton signals in the ¹H NMR spectrum, two active hydrogen protons were found with ¹H NMR chemical shifts at δ 6.15 ppm (O–H) and δ 8.62 ppm (N–H). Due to proton exchange, these two active hydrogen protons disappeared with a little D₂O added. The obtained signals in the range of δ 4.26 to 4.47 and δ 4.58 to 4.73 ppm corresponding to 3-H and 4-H respectively were multiplets, which seems inconsistent with the general split law due to the fluorine atom coupling with 3-H and 4-H. The 5-H (δ 5.00 ppm) signals were doublet of doublet due to the coupling with 4-H and O–H, which changed into a double signal when the hydrogen–deuterium exchanged. The 2-H was in a lower frequency (δ 6.45 ppm) compared with 4-H due to the deshielding effect of chlorine atoms and oxygen atoms. The 7-H and 11-H are chemically equivalent and have the same chemical shift (δ 7.61 ppm). The 8-H and 10-H are chemically equivalent as well (δ 7.85 ppm).

Table 2 The ¹H NMR and ¹H-¹H COSY data of FFC

1H chemical shifts δH (× 10−6) ppm	Multiplicity	Proton number	Coupling constant (Hz)	Assignment	1H-1H COSY δH (× 10−6) ppm
3.15	s	3	–	12-H	–
4.26–4.47	m, dd	2	7.2, 9.2	4-H, 3a-H	4.58–4.73 5.00, 8.62
4.58–4.73	dd, dd	1	5.2, 8.8 5.2, 9.2	3b-H	4.26–4.47
5.00	dd	1	4.0, 7.6	5-H	4.26–4.47, 6.15
6.15	d	1	4.4	OH	5.00
6.45	s	1	–	2-H	–
7.61	d	2	8.0	7-H, 11-H	7.85
7.85	d	2	8.0	8-H, 10-H	7.61
8.62	d	1	8.8	NH	4.26–4.47

Abbreviation: FFC, florfenicol.

The ¹³C NMR spectrum elucidation is shown in [Table 3]. There were 12 carbon signals shown in ¹³C NMR in addition to the solvent peak, among which one primary carbon, six tertiary carbon, two secondary carbon, and three quaternary carbon atoms were found through the comparison among the DEPT-135 and full decoupling ¹³C NMR. For the symmetry of the structure, the signal at δ 43.59 ppm in the highest frequency was assigned to 12-C and the signal at δ 163.41 ppm in the lowest frequency was assigned to the carbon in the carbonyl. The 3-C (δ 81.31, 83.00 ppm, ¹ J _CF = 168.4), 4-C (δ 54.51, 54.71 ppm, ² J _CF = 19.8), and 5-C (δ 69.28, 69.34 ppm, ³ J _CF = 5.7) exhibited a double peak due to the coupling with the fluorine atom by their coupling with 3-H, 4-H, and 5-H in gHSQC. The 2-C at δ 66.20 ppm was in a lower frequency compared with 4-C due to the deshielding effect of chlorine atoms and oxygen atoms. The signal at δ 126.26 ppm was assigned to 8-C and 10-C, and the 7-C and 11-C had the same chemical shift in the δ 126.92 ppm. The 9-C at δ 139.32 ppm could be confirmed by its coupling with 12-H in gHMBC. The signal at δ 147.61 ppm was assigned to 6-C by its coupling with 5-H in gHMBC.

Table 3 The ¹³C NMR, DEPT, HSQC, and HMBC data of FFC

13C chemical shifts δC (× 10−6) ppm	Carbon type	Carbon number	Assignment	HSQC δH (× 10−6) ppm	HMBC δH (× 10−6) ppm
43.59	CH3	1	12-C	3.15	–
54.51, 54.71	CH	1	4-C	4.26–4.47	4.26–4.47, 4.58–4.73, 6.15, 8.62
66.20	CH	1	2-C	6.45	–
69.28, 69.34	CH	1	5-C	5.00	4.26–4.47, 6.15, 7.61
81.31, 83.00	CH2	1	3-C	4.26–4.47, 4.58–4.73	4.58–4.73, 4.26–4.47
126.26	CH	2	8-C, 10-C	7.85	7.61, 7.85
126.92	CH	2	7-C, 11-C	7.61	5.00, 7.61, 7.85
139.32	C	1	9-C	–	3.15, 7.61, 7.85
147.61	C	1	6-C	–	5.00, 6.15, 7.61, 7.85
163.41	C	1	1-C	–	4.26–4.47, 6.45, 8.62

Abbreviation: FFC, florfenicol.

Conclusion

In this study, we elucidated the structure of FFC and demonstrated that different types of solvents made obvious changes in their chemical shifts, especially on 2-H, 3-H, 5-H, and active protons. The clearer ¹H NMR spectrum was obtained when using CDCl₃ as the solvent, but the poor solubility of FFC in it made it difficult to obtain its ¹³C NMR and 2D spectra. This is due to the molecular structure of FFC containing several polar groups, such as amide bond, –Cl, and –F, which have strong polarity, so it is difficult to dissolve in the weak polar solvent of CDCl₃. Therefore, DMSO-d ₆ was eventually used for all the NMR spectra. Different concentrations of FFC in DMSO-d ₆ at different temperatures were used to further evaluate the changes of ¹H NMR chemical shifts and no significant effects were found except for the active protons. As the temperature increases, the chemical shift of active protons gradually decreases because –OH and –NH form hydrogen bonds to a lesser extent at higher temperatures, and the chemical shift is shifted to a higher field. This investigation provided reasonable reference data for further research, especially the choice of the appropriate solvent and temperature, which can lead to significant changes in the ¹H NMR chemical shifts, signal separation, and spin–spin coupling constants.

NMR technology has the advantages of no damage, continuity, high resolution, and strong selectivity, and plays a significant role in compound structure identification, content detection, and dynamic tracking of the chemical reaction process. In future development, NMR can be combined with other analytical equipment, for digital intelligence, and more widely used in various fields.

Conflict of Interest

None declared.

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Publication History

Received: 29 June 2023

Accepted: 31 October 2023

Article published online:
01 December 2023