Florfenicol and Florfenicol Amine Quantification in Bull Serum and Seminal Plasma by a Single Validated UHPLC-MS/MS Method

Florfenicol is a broad-spectrum antibiotic belonging to the amphenicols class that inhibits protein synthesis by binding to bacteria's ribosomal subunits. This drug is commonly used in veterinary medicine to treat bacterial infectious diseases in cattle, swine, poultry, and fish. The proposed method uses a quick protein precipitation with acetonitrile for the extraction of florfenicol and florfenicol amine in serum and seminal plasma, followed by analysis in UHPLC-MS/MS for their simultaneous quantification. A BEH C18 reversed-phase column was chosen for analyte separation, allowing to obtaining sharp and symmetrical peak shapes in a chromatographic run of just 3.5 min under programmed conditions. Two specific transitions were observed for each analyte, and florfenicol-d3 was used as the internal standard. The approach was fully validated in each matrix over ranges suitable for field concentrations of florfenicol and florfenicol amine, showing good linearity during each day of testing (R2 always >0.99). Excellent accuracy and precision were demonstrated, for both analytes, by calculated bias always within ±15% and CV% always below 15% at all QC levels tested. The satisfactory outcomes obtained during recovery, matrix effect, and process efficiency investigations in serum and seminal plasma confirmed the strength of the method for the quantification of target compounds. To our knowledge, this is the first LC-MS/MS-validated approach for the quantification of florfenicol and florfenicol amine in serum and seminal plasma and was successfully applied for the determination of their concentration-time profiles in bulls. This paves the way to understanding the pharmacokinetics of this antibiotic and its active metabolite in bull's seminal plasma, which will enable the design of more appropriate treatment protocols.


Introduction
Florfenicol (FF) is a synthetic antibiotic belonging to amphenicol that acts as an inhibitor of protein synthesis through binding to the ribosomal subunits of bacteria. It shows a broad-spectrum activity against both Gramnegative and Gram-positive organisms, as well as mycoplasma and all organisms sensitive to chloramphenicol [1,2]. FF was frst approved for use in veterinary medicine in the European Union in 1995 as a feed or water additive for the treatment of bacterial infectious diseases in cattle, swine, chicken, dog, cat, and fsh [2][3][4]. In particular, FF is commonly used in cases of bovine respiratory disease (BRD) in cattle, associated with Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Tis antibiotic is also used to treat bovine interdigital phlegmon and bovine keratoconjunctivitis [2].
Its lipophilicity allows FF to cross various anatomic barriers and achieve therapeutic concentrations against intracellular pathogens. For instance, in cattle, it can cross the blood-brain barrier up to 46% [5]. Te half-life of FF in cattle is relatively short when administered intravenously (IV) but increases signifcantly after intramuscular (IM) and subcutaneous (SC) injection [2]. FF is primarily metabolized in the edible tissues of cattle, pig, chicken and fsh, generating forfenicol amine (FFA). Tis happens through different bioconversion pathways, involving intermediate metabolites as forfenicol alcohol (FFOH), forfenicol oxamic acid (FCOOH), and monochloroforfenicol (FFCl) [6,7], as illustrated in Figure 1. Since FFA represents about 35% of the parent drug plasma concentration, it is considered the marker residue of FF by various international legislations, and maximum residual limits (MRLs) have been established for both compounds in all food producing animals [8].
In recent years, several analytical methods based on high-performance liquid chromatography (HPLC) [7,9], high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) [3,[10][11][12][13], gas chromatography (GC) [14], and gas chromatography-mass spectrometry (GC-MS) [15] have been reported for the determination of FF and its metabolites in water, feed, and animal-derived food. On the other hand, the analysis of animal biological fuids has been less frequent, mainly involving serum [16], plasma [13,17], synovial fuid [18], and cerebrospinal fuid [19], but no study has focused on the pharmacokinetics of FF and FFA in seminal plasma. Since no information is available about their distribution in the genital tract, which would allow us to defne the most correct treatment protocols, our research aimed to develop and validate a simple and quick approach to be applied to serum and seminal plasma samples collected during a pharmacokinetic study in bulls. As far as we know, a single method for FF and FFA quantifcation by LC-MS/MS in both matrices has never been proposed.

Chemicals and Reagents.
Analytical standards of forfenicol (molecular weight: 358.21 g/mol; purity: 99.10%) and forfenicol amine (molecular weight: 247.29 g/mol; purity: 97.97%) were purchased from Dr. Ehrenstorfer (Augsburg, Germany); forfenicol-d3 (molecular weight: 361.23 g/mol; purity: 98.5%) was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). Acetonitrile, methanol, and ultra-pure water (all of LC-MS grade) were obtained from Merck (Milano, Italy). Before the start of the study, drug-free serum and seminal plasma samples were collected from healthy bulls and made available to the analytical laboratory for method development.

Standard
Solutions. Stock solutions of FF and FFA at 1,000 μg/mL were prepared by dissolving 10 mg of pure powder of each compound in a 10 mL volumetric fask containing methanol. Florfenicol-d3 (FF-d3) solution at 100 μg/mL, which used as an internal standard (IS), was prepared by dissolving 1 mg of pure powder in a 10 mL volumetric fask containing methanol. Working solutions of forfenicol and forfenicol amine to be used for calibration and quality control (QC) samples were obtained by serial dilution of the stock solutions in acetonitrile and protected from light. All stock solutions were stored at −20 ± 2°C in the dark, and the stability of the three compounds over 1 month of storage was assessed.

Sample Preparation.
All serum and seminal plasma samples were thawed at room temperature (20°C) and prepared using the technique described by Barbarossa et al. [20], with slight modifcations. Briefy, 100 µL of sample and 20 μL of internal standard working solution (FF-d3 at 2 μg/ mL in acetonitrile) were transferred into a 1.5 mL Eppendorf microtube. Ten, 80 μL of acetonitrile was added, and protein precipitation was carried out by vortex mixing for 30 s and centrifuging at 21,000 ×g for 10 min at 20°C. Finally, 20 μL of the supernatant was transferred into a LC glass vial containing 180 μL of ultra-pure water.

2.4.
Liquid Chromatography-Mass Spectrometry. Ultra-high-performance liquid chromatography (UHPLC) was performed on a Waters Acquity UPLC ® system equipped with a binary pump, thermostated autosampler, column oven, vacuum degasser, and condenser (Waters, Milford, MA, USA). Chromatographic separation was obtained with a Waters Acquity BEH C18 (50 × 2.1 mm, 1.7 µm) column coupled with the relative VanGuard precolumn (Waters, Milford, MA, USA) and maintained at 30°C. A gradient program was optimized using a mixture of ultra-pure water (A) and acetonitrile (B) at 0.3 mL/min, switching from 95 : 5 (V A : V B ) to 5 : 95 during the frst 1.30 min, kept for 1.20 min, then back to 95 : 5 over 0.50 min, and fnally re-equilibrated for 0.50 min before the following injection (total runtime 3.5 min). Samples were kept in the autosampler at 20°C, and 7.5 μL from each vial was fnally injected.
Te detector was a Waters XEVO TQ-S microtriple quadrupole mass spectrometer (Waters, Milford, MA, USA), equipped with an electrospray ionization (ESI) source and operating in multiple reaction monitoring (MRM) mode. Capillary voltage was set at −2.80 kV for FF and FF-d3, and at +3.25 kV for FFA. Source and desolvation temperatures were 150 and 600°C, respectively. Cone gas was set at 50 L/h and desolvation gas at 900 L/h; argon was used as a collision gas. Te analyte-dependent MS/MS parameters were optimized through combined infusion of a standard solution of each analyte and the LC mobile phase into the mass spectrometer. Te most abundant transitions identifed for FF, FFA, and FF-d3 are reported in Table 1 with their relative cone voltage and collision energy values.
Data acquisition and analysis were performed using MassLynx 4.2 software (Waters, Milford, MA, USA).

Validation.
Te technique was validated for each analyte following the European Medicines Agency ICH M10 guideline on bioanalytical method validation and study sample analysis [21] during three separated days of testing on serum and seminal plasma. Te validation parameters considered included selectivity, calibration range, accuracy, precision (CV%), extraction recovery (RE), matrix efect (ME), process efciency (PE), carry-over, stability, and reinjection reproducibility.

Selectivity.
After optimizing the chromatographic conditions, the retention time of FF, FFA, and FF-d3 was determined by injecting individual pure solutions at 0.01 μg/ mL. Te selectivity of the method was assessed analysing ten blank bull serum and ten seminal plasma samples to verify the absence of chromatographic signals at the same elution time of FF, FFA, and FF-d3.

Calibration Range.
Matrix-matched calibration curves with a blank sample, a zero sample (blank sample spiked with IS), and calibrators at eight concentration levels were freshly prepared in both matrices in separate sessions following the procedure described in the sample preparation section (adding 20 µL of FF or FFA spiking solution in acetonitrile, and then 60 µL of acetonitrile). Te calibration range (LLOQ-ULOQ) was 0.05-10 μg/mL for FF in both serum and seminal plasma, 0.002-200 μg/mL for FFA in serum, and 0.005-1000 μg/mL for FFA in seminal plasma. Te concentrations of all the calibrators for each curve are reported in Table 2. Peak area ratios between FF or FFA and the internal standard FF-d3 were plotted against their concentration, and a linear least squares regression model was applied. Te accuracy of all the calibration standards should be within ±20% of the expected concentration at the LLOQ and below ±15% at all the other levels, and the resulting correlation coefcient (R 2 ) was considered acceptable if ≥ 0.99. All calibrators had to produce chromatograms with a signal-to-noise (S/N) ratio >10.

Accuracy and Precision.
To evaluate the intra-and interday accuracy and precision of the method, quality control (QC) samples at four diferent concentrations (shown in Table 2) were prepared in 5 replicates along with each calibration curve. Accuracy, expressed as the relative diference between measured value and expected concentration, was evaluated at each QC level and considered acceptable if within ±15% the nominal concentration (±20% at the LLOQ). Similarly, precision, defned as the coefcient of variation (CV%) among repeated individual measures, had to be <15% (<20% at the LLOQ) for each QC level.

Extraction Recovery, Matrix Efect, and Process
Efciency. Te potential matrix efect was frst verifed by the postcolumn infusion technique: during the injection of  Note: the product ion used for quantifcation is in bold.
a blank matrix samples in the LC-MS/MS system, standard solutions of each compound at 0.5 µg/mL were coinfused in the MS interface to evaluate the stability of the produced signal.
An evaluation of RE, ME, and PE was performed following the approach described by Matuszewski et al. [22], in which peak areas obtained from three types of samples are compared: (A) Standard calibrators in mobile phase, containing the same amount of FF or FFA as the third QC level (1 µg/mL of FF and 0.02 µg/mL of FFA in serum; 1 µg/mL of FF and 0.1 µg/mL of FFA in seminal plasma); (B) blank samples of each matrix extracted as described above and added with the same amount of analyte; and (C) samples of each matrix fortifed with the same amount of analyte and extracted as described above. Tree replicates of each type of samples were prepared, using drug-free matrices collected from three diferent animals, in order to also assess possible subject-related diferences. Te following formulas were used to compare the three types of samples and evaluate RE, ME, and PE: (1)

Carry-Over.
For each matrix and analyte, blank samples were analysed immediately after the injection of the ULOQ (10 μg/mL for FF in serum and seminal plasma; 0.2 μg/mL for FFA in serum, and 1 μg/mL for FFA in seminal plasma) to assess the absence of residual analyte.

Stability and Reinjection
Reproducibility. Diferent tests were performed to assess the stability of target analytes in the two matrices and in processed samples. Te longterm stability of each analyte in serum and seminal plasma kept in the freezer (−20°C) was evaluated by preparing additional QCs (lowest and highest level, n � 3) to be analysed after 1 month of storage. Te mean concentration at each level had to be within ±15% of the nominal concentration.
Te stability of processed samples was frst investigated by reinjecting the lowest and highest QCs (n � 5) from the frst day of validation after being left in the autosampler (20°C) for 24 h. Similarly, QCs from the second and third days of validation were frozen (−20°C) after analysis, then thawed and reinjected after 48 h and 7 days, respectively, to assess reinjection reproducibility. For each series of samples, the mean concentration at each level had to be within ±15% of the nominal value.
Te calculation of the arithmetic means of repeated samples and of the abovementioned validation parameters was performed using the Microsoft Excel software.

Application of the Method.
Te proposed method was developed to investigate the trends of forfenicol and forfenicol amine concentrations in bull serum and seminal plasma, providing novel information for optimized dosage regimens of this antibiotic. Te suitability of this approach was assessed by analysing a preliminary series of samples collected at diferent timepoints (0, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h) from a clinical and subclinical healthy bull (Hereford, 17 months, 331 kg) administered with FF at 20 mg/kg through IM injection in the neck. Semen was collected from the bull by electroejaculation using an electro-ejaculator in automatic mode with a two-electrode rectal probe of 60 mm diameter (Pulsator V, Lane Manufacturing, Denver, CO, USA). All the samples were immediately refrigerated at 4°C, then centrifuged for 30 min at 600 ×g and stored at −80°C within the frst hour. Te samples were transported to the UHPLC-MS/MS laboratory under controlled conditions, maintaining a temperature of −20°C throughout the shipment, and stored at −80°C upon arrival. Procedures used in the in vivo experiment were performed according to the standards for the "Use of Animals in Research and Education" by the World Organization for Animal Health (OIE) [23] and approved by the 3R Ranch owners.

Method Development.
Te method presented here was validated according to the current European Medicines Agency guidelines on bioanalytical method validation [21] and is capable of determining forfenicol and forfenicol amine concentrations in serum and seminal plasma. To the best of our knowledge, this is the frst validated approach described for the quantifcation of FF and FFA in either biological fuid using UHPLC-MS/MS.

Veterinary Medicine International
Diferent stationary phases (BEH C18 and HSS T3), mobile phase compositions (water and acetonitrile or methanol, with or without pH modifers), and gradient eluent conditions were tested to optimize chromatography. Te best resolution, peak shape, and intensity signal for FF, FFA, and FF-d3 was obtained on a BEH C18 column using a programmed combination of water and acetonitrile without any additive. Tese conditions resulted in optimal conditions also considering that forfenicol and forfenicol-d3 require negative electrospray ionization (ESI−), while forfenicol amine only produces detectable MS signals when positively charged (ESI+). With this setup, analysis can be carried out in a 3.5 min run, allowing the processing of even large batches of samples in a relatively short time.
A consistent part of the methods described in the literature employ organic solvents such as ethyl acetate [24,25], acetone, dichloromethane [26], and acetonitrile with or without formic acid [27], or alkaline pH conditions for FF extraction from diferent animal tissues or feed. Other published applications for its quantifcation in plasma and serum are based on multiple liquid-liquid and/or solid-phase extraction techniques [16,17]. We managed to avoid such expensive and time-consuming approaches, adapting a sample preparation procedure for tulathromycin quantifcation in plasma, seminal plasma, and urine previously validated by our Veterinary Medicine International group [20]. Moreover, compared to previous studies, the present method reduces sample and organic solvent volumes to just 100 µL and bypasses the fnal fltration step. Te lower amount of matrix required for analysis makes the approach suitable for PK studies, where repeated sample collection is necessary. Te simple and quick sample treatment, consisting of protein precipitation and dilution of the sample, is a further strength point of this method, allowing it to process 24 samples in less than 15 min. Furthermore, FFA was not included in previous analytical applications on blood matrices [16][17][18]. In order to measure FF and FFA at levels comparable to those found in real serum and seminal plasma samples, diferent vial dilution factors were tested, and a twenty-fold dilution was fnally chosen.

Method Validation.
Te injection of pure standards of forfenicol, forfenicol amine, and forfenicol-d3 allowed us to defne their retention times, which were 1.28, 1.24, and 1.28 min, respectively. For each matrix and analyte, the analysis of ten blank samples did not show chromatographic interferences at the retention time of the monitored transitions, proving the good selectivity of the method, as shown in Figure 2.
In all the calibration curves analysed, the coefcient of determination (R 2 ) was always ≥0.99, and calibration standards was always within ±15% of the nominal value which confrmed the linearity of the method over the specifc ranges of concentration. Accuracy and precision were always within ±15% and <15%, respectively, at all QC levels in intraday and interday conditions (data are reported in Table 3).
During the postcolumn infusion test, no ionization suppression or enhancement were observed in the monitored transitions around the retention time of target analytes, giving a frst demonstration of the absence of the matrix efect in both serum and seminal plasma. Te chromatographic signals obtained for each analyte are shown in Figure 3. Te analysis of standard calibrators in the mobile phase, blank matrix samples spiked before extraction, and blank matrix samples spiked after extraction confrmed the absence of any signifcant matrix efect, as well as the optimal recovery and global process efciency. A complete summary of matrix efect (ME), recovery (RE), and process efciency (PE) data is shown in Table 4. No carryover was observed injecting blank samples following the highest point of each calibration curve. Te stability of target analytes in each matrix was assessed after 1 month at −20°C, and the average response remained always within ±8% of the initial value. Similarly, samples reinjected after being left for 24 h in the autosampler at 20°C or stored at −20°C for 48 h and 7 days did not show any relevant variation.

Application of the Method.
Te method was successfully applied to serum and seminal plasma samples collected during the evaluation of the pharmacokinetic profles of FF and FFA in one healthy bull following IM administration of FF at 20 mg/kg. Te obtained concentration vs. time curves are shown in Figure 4, proving that the measurement range of this approach is consistent with the levels of target analytes found in the two matrices. Tis investigation provided interesting information on the behaviour of this antibiotic and its active metabolite not just in serum but also in a novel matrix such as seminal plasma. Te results showed higher levels of FF and FFA in seminal plasma than in serum and highlighted how they can both be found at relevant   Veterinary Medicine International concentrations even 7 days after administration. Further confrmation of their long-lasting concentrations in seminal plasma will be critical to design appropriate treatment protocols (dose, route, and frequency), also in relation to specifc MIC values associated with genital tract infections in bull.

Conclusions
Te present work describes, for the frst time, a single UHPLC-MS/MS technique for the quantifcation of forfenicol and its main metabolite, forfenicol amine, in bull serum and seminal plasma. Te method combined an easy and fast laboratory procedure with optimal analytical performance. Te validated ranges of concentrations were suitable for the detected levels of target analytes in each matrix and gave a frst insight into their pharmacokinetics. Te application of the technique to a larger number of patients will allow us to calculate the main PK parameters of FF and FFA and further investigate their behaviour in these biological fuids.

Data Availability
Te data used in this study are available upon reasonable request to the corresponding author.

Conflicts of Interest
Te authors declare that they have no conficts of interest.