Gentamicin is a broad-spectrum bactericidal aminoglycoside antibiotic, produced by fermentation of Micromonospora purpura or M. echinospora. It is effective against wide variety of serious bacterial infections caused by susceptible gram-negative and some grampositive aerobic bacteria [1,2]. Gentamicin is not a uni-molecule but a complex mixture of four major components, designated as C₁, C₂, C_1a, C_2a, and minor ones like C_2b. The components differ in their degree of methylation on the purpursamine ring [1].

It has been recognized that there is a wide variation in the major component ratio between different pharmaceutical gentamicin preparations [3,4] and therefore, the composition of the final product can vary considerably. Proportions of the different components in most commercial preparations fall within limits that are set and mentioned by the US pharmacopoeia [5] is 25-50% for C₁, 10-35% for C_1a and for sum of C₂ and C_2a are 25-55%. The British pharmacopoeia [6] limits are 25-50% for C₁, 15-40% for C_1a, and 20-50% for sum of gentamicin C₂ and C_2a. European Pharmacopoeia [7] determines the amount of C₁, C_1a and the sum of C₂ and C_2a were limited to 20-35, 10-30 and 40-60%, respectively.

Nephrotoxicity and ototoxicity are the most common side effects associated with the use of gentamicin. The severity of toxicity can vary depending on whether a single or multiple-daily administration plan is used [8]. Moreover, the available data reported remarkable differences in nephrotoxicity for gentamicin components in animals [9]. Consequently, the correlation between toxicity and pharmacokinetics of gentamicin components is important. Gentamicin C₁ has different disposition kinetics than the gentamicin complex when given separately to patients [10].

Several methods have been developed for determination of gentamicin. Only chromatographic methods are capable of identifying and quantifying the individual components of the gentamicin complex. Gentamicin has no UV or visible absorbing chromophores and therefore cannot be detected by traditional techniques without derivatisation [11,12]. This necessitates gentamicin derivatization to allow its detection with the required sensitivity. Either pre- or post- column derivatisation for fluorescence or UV detection can be implemented in this process. Gentamicin has been derivatised previously with O-phthalaldehyde (OPA), dansyl chloride, fluorescamine, 9-fluorenylmethyl chloroformate (FMOC-CI), 1-fluoro-2, 4-dinitrobenzene (DNFB) and 2,4,6-trinotrobenzenesulfonic acid (TNBS) [11].

The pharmacokinetics of individual gentamicin components were studied in dogs [13], turkeys [14] and the horse [15] after intravenous administration only. However, there is no available pharmacokinetics data in other species of animals including chicken. The purpose of this study was to determine and calculate the pharmacokinetics and bioavailability of four major gentamicin components in chicken plasma by different route of administration (IV, IM, SC and oral), using reversed-phase high performance liquid chromatography (RPHPLC) and pre-column derivatization with phenylisocyanate (PIC).

Experimental animals

Fifty 2-2.5 kg body weight (bw) broiler chickens (Hubbard x Hubbard) of 40-45 days old were used in this study. These chickens were purchased from a local poultry farm at 3 weeks old. They were placed in the Animal House at Jordan University of Science and Technology. The animals were monitored for 2 weeks for any apparent clinical signs and to ensure that they are free from antibiotics before drug administration. The Animal House temperature was maintained at 25 ± 2°C and humidity at 45-65%. All chickens had free access to water and antibacterial-free food.

Drug

Authentic standard powder of gentamicin sulphate with known amounts of gentamicin components (33, 25.5, 22 and 19.5% for C_1a, C₁, C_2a and C₂, respectively) was provided by North China Pharmaceutical Group (Hualuan Co. Ltd, Shijiazhuang, China), batch no. (040537). The drug (300 mg) was dissolved in sterile distilled water to a total volume of 15 ml to give a final concentration of 20 mg/ml prior drug administration.

Peak assignment

Peak assignments were made by elution of gentamicin sulphate standard solutions. The relative proportion described from the standard compositions has permitted peak identification. Gentamicin C_1a, C₁, C_2a and C₂ were identified by comparison of their retention time with those identified previously [16].

Chemicals and reagents

All the chemicals employed were of analytical grade. Acetonitrile and water were HPLC-grade (Frutarom, UK), Trifluoroacetic acid (TFA) and Triethylamine (TEA) were purchased from Scharlau, Spain. Phenylisocyanate (PIC) was purchased from Merck, Germany.

HPLC system

Chromatography was carried out on binary high pressure HPLC system (Shimadzu, Japan) which consisted of LC-10A DVP HPLC pump, SIL-10A DVP auto injector, SPD-10 AVP UV-vis detector, SCL-10 AVP system controller, DGV-12 A degasser, Shimadzu class- VP software Ver 6.12 SP4. Chromatographic separation was performed using Chromolith RP-18e (4.6 mm i.d. × 50 mm length, macropore 2 μm, mesopore 2 nm, Merck, Germany).

Experimental design

Chickens were individually weighed before drug administration and doses were calculated accordingly. The chickens were divided into 5 equal groups (10 chickens / group) in a parallel design. Chickens of group 1 did not receive any drug and served as a control group. Chickens of group 2, 3, 4 and 5 were received total gentamicin (5 mg/kg) body weight as a single IV, IM, SC and oral administration, respectively. Gentamicin was given in the right brachial vein, pectoral muscle, under the skin of the neck and directly by thin plastic syringe into the crop for IV, IM, SC and oral administration, respectively. Food was withheld for 12 h before drug administration and was offered 6 h after drug administration to exclude any influence of feed on the absorption of the drug. Water was given freely to all groups. Blood samples (1- 1.5 ml) were collected from the left brachial vein into heparinized tubes at 0 (pretreatment), 5, 15 and 30 min and 1, 2, 4, 6, 8, 12, 24 and 48 h after drug administration. The samples were directly centrifuged at ~1000 g for 10 min to obtain clear plasma and stored at –20°C until analysis.

Preparation of standard curve

Daily fresh calibration curves were prepared by dissolving dried gentamicin mixture powder in HPLC-grade water, in a measuring flask to obtain a concentration of 1000 μg/ml. The stock solution was added to HPLC-grade water or chicken plasma to produce 1, 10, 25, 50 and 100 μg/ml. Four calibration curves were made for each concentration according to their ratios. The calibration curves were obtained by plotting the peak height as a function of the respective concentrations for each component and the linear regression was calculated for each component.

Sample preparation

Concentrations of gentamicin components in plasma were assayed according to previously described method [16] with slight modification. The calibration and plasma samples of gentamicin were prepared by adding 200 μl of plasma to 300 μl of triethylamine solution (5 mg/ml in 90% acetonitrile and 10% water) to precipitate plasma protein. The mixture was shaken for 15 second by vortex mixer and centrifuged at ~1000 g for 5 min. The supernatant (400 μl) was transferred to clean glass tubes and 200 μl of phenylisocyanate (5 mg/ml in acetonitril) was added as derivatizing agent. The mixture was shaken by vortex mixer for 10 seconds and transferred to a shaker water-bath. The water path was set at 65°C for 30 min with slow shaking. The mixture was then transferred to Eppindorf tubes and centrifuged at ~1000 g for 5 min to ensure complete precipitation and clearness of the resultant. The clear supernatant was injected directly into the HPLC system using special glass vials.

Chromatographic condition

Gentamicin was eluted with a mobile phase consisting of acetonitrile-water (36:64, v/v) and 0.1% of triflouroacetic acid was added in aqueous acetonitrile. The mobile phase was filtered through a 0.45 μm membrane filter and degassed. The flow rate was performed at 2 ml /min and the UV detector set at a wavelength of 240 nm. The volume of injection was 100 μl.

HPLC methods for gentamicin C_1a, C_2a, C₁ and C₂ were validated by measuring the specificity, accuracy, precision, linearity, sensitivity and recovery in chicken plasma. The specificity of this method was assured since there were no interfering peaks present in chromatograms corresponding to the retention time of gentamicin –PIC derivatives. The accuracy of the method was 98.6, 100.2, 99 and 99.2% for gentamicin C_1a, C_2a, C₁ and C₂, respectively. The intra-day coefficients of variation (CV) for 4 major components ranged from 3.2 to 6, whereas, the interday CV ranged from 2 to 7. The limit of quantification (LOQ) was 0.3, 0.25, 0.25 and 0.2 μg/ml for gentamicin C_1a, C_2a, C₁ and C₂, respectively based on signal-to-noise ratio of 6:1. Moreover the mean percentage recoveries of gentamicin C_1a, C_2a, C₁ and C₂ from the plasma were 93%, 95%, 94% and 98%, respectively. Gentamicin derivatives C_1a, C_2a, C₁ and C₂ were stable during 24 h from preparation.

Pharmacokinetics analysis

The pharmacokinetic analysis of the data was performed using non-compartmental analysis based on statistical moment theory (SMT) according to previously described methods (Gibaldi and Perrier, 1982), with the help of a commercially available software program (WinNonlin^®, Pharsight Corporation, Cary, NC, USA). The parameters calculated were: area under plasma concentrationtime curve (AUC) using linear trapezoid method; area under the first moment curve (AUMC); mean residence time (MRT), where MRT= AUMC/AUC; volume of distribution (Vd_area), where Vd_area=(dose/ AUC × β); total body clearance (CLB), where CLB = dose/AUC; and apparent volume of distribution at steady state (V_ss), where V_ss=MRT × CLB. The absolute bioavailability (F) was calculated as (AUCnon IV/ AUCIV) ×100. The maximum plasma concentration (C_max) and time to maximum concentration (T_max) following extravascular administration were determined empirically directly from the time-concentration curve. Doses of the individual components were calculated by multiplying the administered dose (5 mg/kg) by the percentage of the component contained in the gentamicin formulation. C_max and AUC_0-∞ were normalized by dose prior to comparison.

Statistical analysis

One-way analysis of variance (ANOVA) was used to test the hypothesis of no differences between the average pharmacokinetic parameter values between the 4 major gentamicin components. If the means were different, a multiple comparison of the means was performed using the Fisher’s Least Significant Difference test (LSD). The data were log-transformed prior to analysis, since they were not normally distributed on a linear scale. Some parameters (C_max and T_max) were not normally distributed, even after log-transformation, and the Kruskall-Wallis ANOVA on ranks and Tukey test were used for these data. The differences were considered significant when P<0.05. All data are expressed as the geometric mean ± SD of the log data, the latter being an approximation of the co-efficient of variation [17]. For C_max and T_max, the median, 25^th percentile and 75^th percentile are reported.

A representative HPLC chromatogram of blank chicken plasma containing PIC and TEA without gentamicin and the separation of the gentamicin components in chicken plasma are illustrated in figures 1A and 1B, respectively. The calibration curves of gentamicin components, spiked in chicken plasma, were linear (data not shown). Close correlation with the linear regression equations were observed for all four components (r₂=0.999, 0.998, 0.998 and 0.997 for C_1a, C_2a, C₁ and C₂, respectively). The peak heights were proportionally related to gentamicin component concentrations.

Figure 1a: A representative HPLC chromatogram of chicken plasma containing containing PIC and TEA without gentamicin.

Figure 1b: A representative HPLC chromatogram of chicken plasma containing gentamicin (C_1a, C_2a, C₁ and C₂) showing the separation.

The mean concentrations ± SE of gentamicin C_1a, C_2a, C₁, C₂ and total gentamicin (determined by summation of the concentrations of 4 major components) after single IV, IM and SC administration of a single dose of gentamicin (5 mg/kg bw) are shown in figures 2, 3 and 4, respectively. The pharmacokinetics parameters of gentamicin C_1a, C_2a, C₁, C₂ and total gentamicin following single IV, IM and SC administration of a single dose of gentamicin (5 mg/kg bw) are shown in tables 1, 2 and 3, respectively.

Figure 2: Semilogarithmic plot showing the plasma concentration-time profile of gentamicin C_1a, C_2a, C₁ and C₂ and total gentamicin after IV (5 mg/ kg bw) administration. Values are mean ± SE (n=10).

Figure 3: Semilograthimic plot showing the plasma concentration-time profile of gentamicin C_1a, C_2a, C₁ and C₂ and total gentamicin after IM (5 mg/ kg bw) administration. Values are mean ± SE (n=10).

Figure 4: Semilograthimic plot showing the plasma concentration-time profile of gentamicin C_1a, C_2a, C₁ and C₂ and total gentamicin after SC (5 mg/ kg bw) administration. Values are mean ± SE (n=10).

Table 1: The pharmacokinetics parameters of gentamicin C_1a, C_2a, C₁, C₂ and total gentamicin following single IV administration. Values are geometric mean ± SE of the log-transformed data (n=10).

Table 2: The pharmacokinetics parameters of gentamicin C_1a, C_2a, C₁, C₂, and total gentamicin following single IM administration.

Table 3: The pharmacokinetics parameters of gentamicin C_1a, C_2a, C₁, C₂, and total gentamicin following single SC administration. Values are mean ± SE (n=10).

The apparent volume of distribution (V_ss and Vd_area) for the C₁ component was significantly smaller than for any of the other components individually or combined. In addition, the t&fraC₁2;β and MRT were significantly shorter for C₁ following intravenous administration (Table 1). The data collected after intramuscular administration also suggests that C₁ has a smaller apparent volume of distribution (Vd_area/F). This is also the most likely reason for C₁ having a significantly higher C_max/Dose following intramuscular administration. All gentamicin components were rapidly and extensively absorbed following intramuscular and subcutaneous administration with the exception of C_1a, which had a bioavailability of 58 and 35%, respectively. This component also had a lower C_max/Dose and AUC_0-∞/Dose as well as a higher Vd_area/F and CLB/F. Gentamicin was not detected in chicken plasma, after a single oral administration of gentamicin (5 mg/kg bw).

Gentamicin is a polarized water-soluble compound; it is excreted un-metabolized via the kidney and has very poor intestinal membrane permeability [12,18]. There are numerous reports of gentamicin pharmacokinetics in human and animals. With few exceptions, these consider gentamicin to be single molecule and describe the pharmacokinetics of total gentamicin only. It is interesting that gentamicin consists from 4 major components (C_1a, C_2a, C₁ and C₂) and other minors. The pharmacokinetics of individual gentamicin components has not been targeted by many researchers, but this does not detract from its importance. On the contrary, the pharmacokinetics of gentamicin components has been a subject of interest due to clinical and toxicological considerations. The study of the individual components has been hampered by the lack of a suitable calibrated method of detection and the analytical problems associated with gentamicin derivatization.

Direct UV detection of gentamicin is not possible because gentamicin has no UV or visible chromophores and cannot be detected by traditional techniques [19,20]. Therefore, derivatization of gentamicin to allow its detection with suitable sensitivity is necessary. Pre-column derivatization with a suitable fluorescent reagent allows for the simplest, accurate and most sensitive analysis. Derivatization of gentamicin by O-phthalaldehyde (OPA) [21], dansyl chloride [21], fluorescamine [22], 9-fluorenylmethyl chloroformate [1], 1-fluoro-2, 4-dinitrobenzene [21] and 2,4,6-trinitrobenzenesulfonic acid [23,24] were reported. However, these reagents are with drawbacks related to reagent stability, time consuming and detection sensitivity. For example, OPA is unstable and the elution of the OPA derivatives has been found to affect by the concentration of inorganic cations in the HPLC mobile phase [19,11].

In our previous study, the concentrations of gentamicin in chicken plasma after IV, IM, SC and oral administration were determined using microbiological assay [25]. This bioassay method is simple and inexpensive, but unable to quantify of the individual components of gentamicin [12,25]. High correlation (r₂=0.97) was found between HPLC and bioassay methods in determining the mean plasma gentamicin in chickens. However, there were significant differences in some pharmacokinetics parameters when HPLC and bioassay were compared [25].

We modified a simple and rapid liquid chromatographic method for determination of gentamicin components in plasma using Phenylisocyanate (PIC) as pre-column derivating reagent. This method shows good specificity, accuracy, stability, precision and linearity. To our knowledge, this is the first study that determined and calculated the pharmacokinetics of each gentamicin component (C_1a, C_2a, C₁ and C₂) separately in chicken plasma using RP-HPLC and phenylisocyanate as derivatizing reagent.

After single IV administration, gentamicin C₁ has the shortest t1/2β (1.69 h) followed by C₂ (3.87), C_2a (5.24 h) and C_1a (6.45 h). These differences are attributable to differences in both clearance and apparent volume of distribution between the components. The estimated value for CLB is highest for C₂ (112.73 ml/hr/kg), followed by 79.60, 79.76 and 83.1 ml/hr/kg for C₁, C_1a and C_2a respectively. In contrast, the estimated value for V_ss is highest for C_1a (539.15 mL/kg) followed by 502.70, 453.50 and 132.42 mL/kg for C_2a, C₂ and C₁ respectively.

After a single IM administration of 5 mg/kg bw of gentamicin the C_max/Dose were 4.48, 2.49 1.94 and 1.73 μg/ml all occurring at 0.5 hours for gentamicin C₁, C_1a, C₂ and C_2a, respectively. The higher C_max/Dose for C₁ can be ascribed to the smaller apparent volume of distribution for this component. The calculated value for Vd_area/F was 304.30, 674.52, 972.63 and 1242.65 ml/kg for components C₁, C_1a, C₂ and C_2a respectively. The shortest t1/2β was noted for gentamicin C₁ (2.67 h), whereas 3.39, 4.70 and 6.61 h were calculated for gentamicin C_1a, C₂ and C_2a respectively. Once again, this can be accounted for by the smaller apparent volume of distribution for this component. Gentamicin C₁ had the highest bioavailability (F=101%), while gentamicin C_1a had the lowest (F=58%). The bioavailabilities of gentamicin C₂ and C_2a were 78 and 64%, respectively.

After a single SC administration of gentamicin at a dose of 5 mg/ kg bw, C_max/Dose were 4.74, 1.79, 2.81 and 2.78 μg/ml at 0.50, 1.00, 0.75 and 0.50 h for gentamicin C₁, C_1a, C₂ and C_2a, respectively. The shortest t1/2β was once again noted for gentamicin C₁ (1.54 h), while gentamicin C_1a, C₂ and C_2a had higher t1/2β values 2.37, 3.32 and 3.54 h, respectively. Gentamicin C_1a had the lowest bioavailability (35%), resulting in a significantly lower C_max/Dose (1.79 μg/ml) compared with the other components. The C_max/Dose was 4.74, 2.81 and 2.78 μg/ml for components C_1a, C₂ and C_2a, respectively.

The extrapolated percentages of the AUC_0-∞ for components C_1a and C_2a were greater than the generally accepted 20% following both intravenous and intramuscular administration (IV: 31% and 25% respectively; IM: 23% and 40%). This may have affected the accuracy with which some of the pharmacokinetic parameters were estimated for these components.

Our results showed significant differences between the 4 major components in some pharmacokinetics parameters after IV, IM and SC administration of gentamicin at a dose of 5 mg/kg bw in broiler chickens. Specifically, the apparent volume of distribution was smaller and the t&fraC₁2;β was shorter for component C₁. Also notable was that C_1a seemed to have been poorly absorbed following SC administration. Differences have also been reported in the pharmacokinetic parameters of the different gentamicin components in dogs (larger apparent volume of distribution and slower clearance of component C₁) [13] and horses (faster clearance of C_1a) [15]. The differences in PK were mainly attributed to higher tissue binding for C₁ compared with others [13]. This supports the hypothesis that there are significant differences in the pharmacokinetics of gentamicin components, but these differences are not consistent across species.

Other study conducted by Shem-Tov et al. [14] found significant differences in the pharmacokinetics profiles between major components in body tissue when the drug was given to turkeys. Therefore, the differences in total gentamicin pharmacokinetics and nephrotoxicity reported in the previous studies may result from the differences in the pharmacokinetics behavior of the different component.

After oral administration of gentamicin at a dose of 5 mg/kg bw, no component could be detected in plasma samples in all tested chickens. The oral bioavailability (F) was 0.0%. These finding are in consistent to those described previously [23]. This may due to high polarity and cationic nature of the drug that result in scant absorption from gastrointestinal tract.

In conclusion, our results showed significant differences in some pharmacokinetics parameters between four gentamicin components (C_1a, C_2a, C₁ and C₂) after administration of single mixture of gentamicin by IV, IM, SC and oral routes. The differences may have clinical and toxicological implications, and could explain the high variation in total gentamicin pharmacokinetics. A modified rapid and simple method was developed for the determination of gentamicin components in chicken plasma. This RP-HPLC method used precolumn derivatization of gentamicin with Phenylisocyanate (PIC). This method is able to measure 4 major components of gentamicin in plasma at low concentrations. It is therefore, well suited for performing pharmacokinetics and analytical studies. Further studies are needed to determine the minimum inhibitory concentration for each gentamicin component against susceptible microorganisms of interest. In addition, toxicological profile for each of these components should be studies in different animal species. Based on these studies, a determined ratio of gentamicin components should be recommended to avoid the wide variation in pharmaceutical preparations.

This study was funded by the Deanship of Scientific Research at Jordan University of Science and Technology (grant no. 121/2004).

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