LC Analysis of Oxytetracycline and Chlortetracycline: Application for In Vitro Bio-Equivalence Study of Veterinary Medicines 2009, 69, 215–220

Francesca Mancini1, Cristina Cavallari1, Paola Filippi2, Lorenzo Rodriguez1, Anna Maria Di Pietra1, Vincenza Andrisano1,& 1

2

Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy; E-Mail: [email protected] Industria Italiana Integratori TREI S.p.A, Viale Corassori 62, 41100 Modena, Italy

Received: 26 June 2008 / Revised: 8 September 2008 / Accepted: 19 September 2008 Online publication: 9 November 2008

Abstract A liquid chromatographic method has been applied for the analysis of two antibiotics widely used in the veterinary field, oxytetracycline dihydrate and chlortetracycline hydrochloride in premixes for medicated feeding stuffs for veterinary use. In particular, the validated method was employed to study the releasing profile of each drug from two formulations, a commercially available and a new formulation, having different excipient composition. The dissolution profiles obtained from the chromatographic analysis allowed to verify the in vitro bio-equivalence of the commercial and the new formulations for oxytetracycline and chlortetracycline.

Keywords Column liquid chromatography Bio-equivalence study Veterinary medicines Oxytetracycline dihydrate and chlortetracycline hydrochloride

Introduction In vitro dissolution has been recognized as an important aspect in drug development. Drug release/dissolution from solid pharmaceutical dosage forms has been the subject of interest of many research

Original DOI: 10.1365/s10337-008-0874-1 0009-5893/09/02

groups both in human and in veterinary field. In the development and production of a new solid dosage form it is necessary to ensure that drug dissolution occurs in order to reach the drug bioavailability [1]. Tetracyclines are natural broadspectrum antibiotics widely used as

veterinary medicine for in feed or in water administration to prevent infectious diseases. Tetracyclines exhibit general poor stability [2] and upon storage in animal feeds and premixes may be subjected to extensive degradation (i.e., epimerisation). Analysis of tetracyclines and their determination in feeds by LC proved to be rather difficult [3]. The chromatographic conditions are a critical part of the LC methods; a variety of stationary phases, such as polymeric reversed phase [4–8], RP-8 [3, 9, 10], RP-18 [3, 11–13] and graphitic carbon [14] columns, have been proposed. In the present work, an LC method for the analysis of oxytetracycline dihydrate and chlortetracycline hydrochloride has been validated. The silica polymeric hybrid stationary phase was found suitable to analyse oxytetracycline and chlortetracycline in veterinary medicines. In particular, the dissolution profiles of the drugs from the correspondent medicated premix were evaluated and in vitro bio-equivalence studies were performed. A commercially available and a new formulation, having a more homogeneous and less powdery excipients, were analysed.

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Experimental Materials Oxytetracycline dihydrate, triethylamine, citric acid monohydrate and orthophosphoric acid (85% v/v) were obtained from Sigma Aldrich (Milan, Italy). Chlortetracycline hydrochloride, commercially available and new medicated premixes, OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200, containing, respectively, 20.6% (w/w) oxytetracycline base and 20.5% (w/w) chlortetracycline base, were provided by Industria Italiana Integratori TREI (Italy). Potassium dihydrogen phosphate (Fluka), dipotassium hydrogen phosphate (Merck) were of high-grade purity. Phenomenex Gemini C18 (150 9 2.0 mm I.D.) and Luna C18 (150 9 4.6 mm I.D.) stationary phases were from Chemtek Analitica (Bologna, Italy). Chromolith C18 performance stationary phase (100 9 4.6 mm I.D.) was from Merck (Darmstadt, Germany). All the other chemicals were of analytical reagent grade (Carlo Erba Reagenti, Milan, Italy) and were used without further purification. Water used for the preparation of solutions and mobile phases was purified by a Milli-Rx apparatus (Millipore, Milford, MA, USA). The buffer solutions were filtered through a 0.45 lm membrane filter and degassed before their use in LC.

Apparatus and Chromatographic Conditions Chromatographic analysis were performed on an LC system consisting of a Jasco PU-1580 solvent delivery system connected to a Jasco autosampler model AS-2055 and a Jasco UV-2070 detector system. UV–Vis detector was set at 353 and 365 nm for oxytetracycline dihydrate and chlortetracycline hydrochloride analysis, respectively. Routine chromatographic analyses of oxytetracycline dihydrate and chlortetracycline hydrochloride were carried out on a Gemini C18 (150 9 2.0 mm I.D., 5 lm), by using a mobile phase consisting of a buffer solution containing 20 mM triethylamine (TEA) phosphate (pH = 2.5) and

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acetonitrile in the volumetric ratio 90/10 (v/v) for oxytetracycline dihydrate and 80/20 (v/v) for chlortetracycline hydrochloride at a flow rate of 0.3 mL min-1. Samples were injected using loop injections (20 lL) and chromatographic analyses were performed at room temperature (20 ± 2.0 °C).

Method Validation Standard Stock Solutions

Standard stock solutions of oxytetracycline dihydrate (0.53 mg mL21) were dissolved in acidic water (HCl 0.01 M) and were stored at 4 °C. Daily, required volumes of each analyte stock solution were diluted in the mobile phase [20 mM triethylamine (TEA) phosphate (pH = 2.5): acetonitrile 90/10 (v/v)] to prepare the standard working solutions. Standard stock solutions of chlortetracycline hydrochloride (1.01 mg mL21) were prepared in acidic water (HCl 1 M)/acetonitrile 50/50 (v/v) and were stored at 4 °C. Daily, required volumes of each analyte stock solution were diluted in the mobile phase [20 mM triethylamine (TEA) phosphate (pH = 2.5): acetonitrile 80/20 (v/v)] to prepare the standard working solutions. Calibration Curves

Working solutions were prepared by diluting the stock solution with the mobile phase to obtain concentrations ranging within 0.021–0.212 mg mL21 of oxytetracycline dihydrate and within 0.010–0.505 mg mL21 of chlortetracycline hydrochloride. Each solution was stirred for about 30 s, sonicated for about 5.0 min and then filtered before the injection into the LC system. Duplicate injections were performed for each solution. For obtaining the calibration graphs, the analyte peak areas were plotted against the correspondent analyte concentrations (mg mL21). Precision

The repeatability of the chromatographic method was evaluated by injecting the same standard solution of each com-

pound (0.202 mg mL-1 oxytetracycline dihydrate and 0.337 mg mL-1 chlortetracycline hydrochloride) three times on the same day under the optimized chromatographic conditions described above. The peak areas were integrated and the standard deviations were calculated. The repeatability of retention times was also evaluated. The reproducibility of the analysis was also evaluated, taking account to the contribution from sample preparation (as weighing, aliquoting and diluting), on three different days. Limit of Detection (LOD)

The method sensitivity was evaluated by progressive dilution of standard mixtures of oxytetracycline dihydrate and chlortetracycline hydrochloride by detecting the signal at a wavelength of 353 and 365 nm respectively.

Sample Analysis A fixed amount of OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200 premixes for medicated feeding stuffs (55.5 mg) was introduced in a volumetric flask containing 50 mL of an aqueous solution of 0.1% ortho-phosphoric acid (v/v) (pH = 2) and stirred mildly for 1 h at 40 °C. Then, the solutions were filtered and diluted three times in the corresponding mobile phase before the injection into the LC system under the optimized chromatographic conditions (as described above). The resulting peak areas were integrated and the corresponding oxytetracycline dihydrate and chlortetracycline hydrochloride concentrations were calculated from the respective calibration graphs (described above).

Dissolution Profiles (Time Course Drug Release) In order to determine oxytetracycline dihydrate and chlortetracycline hydrochloride release from the new and old formulations, the dissolution apparatus described in the European Pharmacopoeia VI edn. [4] was employed. The dissolution test was carried out in an aqueous solution of 0.1%

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Results and Discussion Chromatographic Conditions The aim of this work was the application of a reversed-phase LC method in the study of the dissolution profile of two veterinary drugs (oxytetracycline dihydrate and chlortetracycline hydrochloride) from their premixes for medicated feeding stuffs (OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200). The first step was the optimization of the chromatographic conditions of the analysis. The complexity of the chromatographic analysis was due to the chelating complex that tetracyclines usually form with metallic ions and their strong interaction with the silanolic groups of the stationary Original

150 recovery% o.f. recovery% n.f.

Recovery %

100

50

0 0

25

50

75

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Time (min) Fig. 1. Dissolution profiles of oxytetracycline from the medicated premix OSSIBIOTIC 200 PREMIX old and new formulations expressed as drug recovery (%) versus time

150 recovery% o.f. recovery% n.f.

Recovery %

ortho-phosphoric acid (v/v) (pH = 2 to simulate swine gastric pH). An amount of 1.00 g of the premix was introduced in the cylindrical vessel of borosilicate glass in a final volume of 900 mL, at 37 °C (to simulate body temperature). Volumes of 1 mL were withdrawn at fixed times (0– 90 min for oxytetracycline dihydrate and 0–60 min for chlortetracycline hydrochloride) and analyzed under the chromatographic conditions described above. The solution volume was reintegrated in the cell after each withdrawal. The dissolution test was repeated three times for the old and new OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200. The dissolution sample (previously withdrawn at fixed times from the vessel) was diluted three times with the mobile phase before the injection into the LC system (chromatographic conditions as above). Each injection was performed in duplicate. The resulting peak areas were used to evaluate the concentration (mg mL-1) of the drug at the time of withdrawal by external standard working solutions from pure reference compounds, taking into account the dilution factor of each analyzed sample. Dissolution profiles were obtained by plotting the amount of released drug versus time (Figs. 1, 2) by using a non-linear regression fit (one phase exponential association equation). The computer program used to analyze these data was GraphPad Prism 4.0 (GraphPad Software Inc.).

100

50

0 0

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20

30

40

50

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Time (min) Fig. 2. Dissolution profiles of chlortetracycline hydrochloride from the medicated premix CLORBIOTIC 200 old and new formulations expressed as drug recovery (%) versus time

phases, leading to tailed peaks. Usually, in the chromatographic analysis of tetracyclines, it is necessary to add oxalic acid in the mobile phase in order to minimize peak tailing. However, in order to carry out impurities identification, oxalic acid was found to interfere in the LC–ESI–MS analysis of tetracyclines in feeds [3, 13]. In the present work, a silica ‘‘end-capped’’ stationary phase formed by hybrid polymeric silica was employed to analyze oxytetracycline dihydrate and chlortetracycline hydrochloride in short analysis time with an acidic mobile phase, suitable for the analysis without the presence of oxalic acid, as previously reported [15– 17]. As a result, under the optimized chromatographic conditions described above, retention times were found to be approximatively 10 min and 8 min for oxytetracycline dihydrate and chlortetracycline hydrochloride, respectively

(chromatograms shown in Figs. 3, 4). The performance of a Gemini C18 column was compared with two other columns (a classical C18 and a monolithic C18 column), by evaluating the chromatographic behavior of oxytetracycline dihydrate. Peak tailing and longer retention times were observed when these columns were employed, at various mobile phase compositions and flow rates (i.e., organic modifiers as acetonitrile and methanol were mixed together with phosphate buffer, sodium sulfate buffer or TEA buffer at various pHs with flow rates ranging from 0.3 to 1.0 mL min-1). The optimized method for oxytetracycline was only slightly modified (volumetric ratio between triethylamine phosphate pH = 2.5/acetonitrile from 90/10 to 80/20) for the analysis of chlortetracycline hydrochloride. Selectivity was evaluated by injecting standard solutions of

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300000 Oxytetracycline Blank

250000

µAbs

200000 150000 100000 50000 0 0

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6

4

8

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Time (min) Fig. 3. A typical chromatogram of oxytetracycline dihydrate (c = 0.202 mg mL-1) obtained on a Gemini C18 column (150 3 2.0 mm I.D.) overlaid to a blank. Chromatographic conditions: mobile phase 20 mM triethylamine phosphate buffer, pH 2.5/acetonitrile 80/20 (v/v), k = 353 nm, flow rate = 0.3 mL min21

150000

µAbs

100000

50000

0 0

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Time (min) Fig. 4. A typical chromatogram of chlortetracycline hydrochloride (c = 0.126 mg mL21) obtained on a Gemini C18 column (150 3 2.0 mm I.D.). Chromatographic conditions: mobile phase 20 mM triethylamine phosphate buffer, pH 2.5/acetonitrile 90/10 (v/v), k = 365 nm, flow rate = 0.3 mL min-1

oxytetracycline and chlortetracycline into the LC system. As shown in Figs. 3 and 4, peaks were completely separated from the interferents present in the solutions. The selectivity of the chromatographic method was granted as a > 1.6 for oxytetracycline and a > 1.1 for chlortetracycline analysis. A chromatogram of the blank sample, with no interfering peaks, is shown in Fig. 3. Thus, the selectivity for the desired drug peaks allowed us to apply the method for the analysis of veterinary medicines.

Method Validation A linear relationship between peak area of the drug (y) and the corresponding

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concentration (x) was attained. The linearity of response was studied on five different standard solutions of both oxytetracycline dihydrate (range 0.021– 0.212 mg mL21) and chlortetracycline hydrochloride (range 0.010–0.505 mg mL21); these ranges resulted suitable to test the linearity at the levels normally observed in the analysis of veterinary medicines. The calibration graphs were constructed by plotting the peak areas (Y) versus the corresponding analyte concentrations (X, mg mL21); the obtained relationships are for oxytetracycline dihydrate, y = 16850758 (±98236) x 13433, r2 = 0.9997 and for chlortetracycline hydrochloride, y = 98025424 (± 533634) x +278186, r2 = 0.9998.

The reported statistical data represent the average correlation coefficient, slope and intercept for each calibration curve obtained on three different days. Good linearity was therefore obtained even in the absence of internal standard. Ninety-five percent confidence intervals of the y-intercepts were found to be -29906 to 3039 for oxytetracycline and -84670 to 641043 for chlortetracycline. The system suitability specifications and tests are parameters that provide information on the behaviour of a chromatographic system. Some of them are theoretical plate numbers (N), the injection precision (expressed as RSD%, n = 6), the capacity factor (k) and retention times repeatability. These parameters were determined and are reported in Table 1. The method sensitivity was evaluated by progressive dilution of standard solution of each drug and evaluating the peak area at their respective retention times. Oxytetracycline dihydrate and chlortetracycline hydrochloride solutions (6.81 ± 2.84 and 4.58 ± 1.64 lg mL21 respectively) provided a signal-to-noise ratio of approximately 3 (limit of detection; LOD). The LOQ (limit of quantitation; S/N = 10) values were 15.14 ± 6.42 lg mL21 for oxytetracycline dihydrate and 12.27 ± 0.23 lg mL21 for chlortetracycline hydrochloride. Chromatographic precision, as repeatability and reproducibility of the method was evaluated by analyzing three times the same standard solution of each drug on the same day and preparing three different standard solutions with the same procedure and at the same concentrations on three different days, under constant conditions of solvent composition, temperature and flow rate. Precision assays (intra-day precision) shown 0.517 and 0.157% as relative standard deviation (RSD%) for oxytetracycline dihydrate and chlortetracycline hydrochloride peak areas, respectively. Inter-day precision was also very satisfactory, with 0.906% (oxytetracycline dihydrate) and 1.210% (chlortetracycline hydrochloride) RSD% (data obtained on three different days). Intermediate precision was determined by evaluating the influence of additional

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random effects such as operator, instruments and days. RSD% were found to be comprised between 0.457 and 3.891%.

Table 1. System suitability data

Oxytetracycline Chlortetracycline

N

Capacity factor (k)

Instrument precision

Rt repeatability

6,240 4,700

5.83 3.91

0.01 < RSD% < 0.38 0.03 < RSD% < 0.71

0.15 < RSD% < 0.60 0.12 < RSD% < 0.98

Sample Analysis Following the procedure reported above, the determination of the two drugs in the formulations were carried out. Specifically, a fixed amount (55 mg) of OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200 premix for medicated feeding stuffs, new and commercially available samples were individually analyzed by stirring the sample mildly for 1 h at 40 °C in 0.1% ortho-phosphoric acid (v/v) (pH = 2), in a 50 mL volumetric flask. The resulting solutions were filtered and subjected to the chromatographic analysis, by following the conditions reported before. Analyte determination was performed by comparison with an appropriate standard solution. The filtration step allowed the quantitative recovery of the analyzed antibiotics. The analyte contents found in the commercially available and in the new formulations were in good agreement with the declared content. The results obtained are reported in Table 2. The experimental conditions were found suitable to extract the drugs from the excipients of the formulations, and to obtain their dissolution in the acid solution. Drug stability in the experimental conditions was also checked by comparison with an appropriate standard solution. Drugs resulted to be stable during the time required for the experiments in the employed acid solutions. In these conditions, the complete recovery of the drug from the medicated premix was obtained.

In Vitro Bio-Equivalence Study The in vitro bio-equivalence study of the formulations differing in the excipient content was performed by using the reported dissolution test for solid dosage form [18]. Both the commercially available OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200 contained liquid paraffin as excipient, which has been Original

Table 2. Results for the analysis of medicated premixes OSSIBIOTIC 200 (old and new formulation) and CLORBIOTIC 200 (old and new formulation) Formulation

Declared (%)

Found (mg mL21)

Found (%)a

RSD (%)

OSSIBIOTIC 200 old OSSIBIOTIC 200 new CLORBIOTIC 200 old CLORBIOTIC 200 new

20.73 20.53 20.61 20.36

0.228 0.234 0.238 0.225

98.07 102.02 103.90 98.70

0.43 2.80 2.20 0.37

The values obtained are the mean of two independent experiments, each performed in duplicate a Relative to the declared content Table 3. Percentage of oxytetracycline dihydrate released after 90 min of dissolution test from OSSIBIOTIC 200 PREMIX (old formulation) and OSSIBIOTIC 200 PREMIX NEW (new formulation) (Q = percentage of the declared content) No. exp.

Q% old formulation

Q% new formulation

1 2 3 Mean ± SEM

83.86% 89.36% 89.27% 87.50% ± 3.15

86.44% 90.53% 88.88% 88.62% ± 2.06

Table 4. Percentage of chlortetracycline hydrochloride released after 60 min of dissolution test from CLORBIOTIC 200 (old formulation) and CLORBIOTIC 200 NEW (new formulation) (Q = percentage of the declared content) No. exp.

Q% old formulation

Q% new formulation

1 2 3 Mean ± SEM

96.38% 95.26% 100.09% 97.24 ± 2.52

99.10% 97.55% 88.88% 98.07 ± 5.51

substituted with soybean oil in the new formulations. These modifications were planned in order to improve powder flowing and obtain less powdery and more homogeneous formulations. Moreover, liquid paraffin, a mixture of liquid saturated hydrocarbons obtained from petroleum, could interfere with tetracycline absorption, while soybean oil is a digestible vegetable food ingredient, allowed for pharmaceutical purposes since its monograph is reported in the European Pharmacopoeia [19]. For these reasons, the releasing profiles of the commercial premixes for medicated

feeding stuffs were compared with the corresponding new formulation, in order to evaluate if the nature and/or the amount of the new excipient (soybean oil) could influence the release of the drug from the premix. The authorized commercial formulations and the new ones were processed in the same way, as described above. The dissolution profiles obtained for OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200 are shown in Figs. 1 and 2, respectively. Oxytetracycline dihydrate release from OSSIBIOTIC 200 PREMIX old and new formulation was obtained in 90 min (Fig. 1), while the

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release of chlortetracycline hydrochloride from CLORBIOTIC 200 required 60 min to be completed (Fig. 2). Time course drug release was performed in triplicate for OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200 (old and new formulation) and the statistical results are reported in Tables 3 and 4. The recovery, plateau value of the exponential curve, was found to be 88% for oxytetracycline dihydrate and 100% for chlortetracycline hydrochloride, while relative standard deviations (RSD%) were comprised in the range 2.06–5.51%. As shown in Figs. 1 and 2, the dissolution profiles of the old and new formulations were found to be comparable and almost completely overlaid, in accordance with the protocol study (less than 10% differences between the old and new formulation). In particular, the differences between the formulations were found to be 1.12 and 0.83% for OSSIBIOTIC 200 PREMIX and CLORBIOTIC 200, respectively. Thus, it is possible to state that the new formulations do not have significant differences when compared to the commercially authorized formulations.

Conclusion The results obtained with this study shown that the proposed liquid chromatographic method was suitable to

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study the dissolution profile of oxytetracycline dihydrate and chlortetracycline hydrochloride from formulations and to compare two different premixes for medicated feeding stuffs for veterinary use. In particular, it was demonstrated that the presence of a different excipient (soybean oil) made the new formulations more homogeneous without affecting the dissolution profiles of oxytetracycline dihydrate and chlortetracycline hydrochloride from their corresponding veterinary medicine. Finally, it is possible to state that in vitro bio-equivalence between the old and the new formulation was demonstrated for both oxytetracycline and chlortetracycline premixes for medicated feeding stuffs.

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