Posted by James
The HPLC methods used for analysis of gentamicin and citrate proved acceptable, and for both methods the validation studies indicated suitable linearity over an acceptable range, as well as suitable accuracy and precision. The specificity of the methods was established by means of forced degradation, and the peaks for the degradation products all had retention times well separated from those of the analytes. There were no changes in peak symmetry or retention times for parent compounds over the validation period. The presence of citrate was shown to not interfere with the analysis of gentamicin, and the presence of gentamicin was shown to not interfere with the analysis of citrate.
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Validation of Citrate Assay
Regression analysis of peak area as a function of concentration for standard solutions of sodium citrate with concentrations ranging from 400 to 3200 ^g/mL demonstrated linearity, and the coefficient of determination (r2) was 0.9996 (n = 15). The 3 sets of solutions at concentrations 800, 1600, and 2400 ^g/mL had an accuracy of 99.61% (n = 9) and a within- day precision, expressed as relative SD, of 1.06% (n = 9). The between-day precision was 0.88% (n = 18). Chromatograms of citrate showed one peak eluting at 11.0 min.
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Posted by James
Validation of Gentamicin AssayRegression analysis of peak area as a function of concentration for standard solutions of gentamicin with concentrations ranging from 80 to 400 ^g/mL demonstrated linearity, and the coefficient of determination (r2) was 0.9924 (n = 15). The 3 sets of solutions at concentrations of 160, 240, and 320 ^g/mL had an accuracy of 101.0% (n = 9) and a within- day precision, expressed as relative SD, of 2.21% (n = 9). The between-day precision was 2.52% (n = 18). Chromatograms of the phenylisocyanate derivatives prepared from the gentamicin standard showed 3 peaks eluting at 5.3, 5.6, and 6.0 min, which represented gentamicin C^ C , and C2.To quantify the gentamicin, the areas of these 3 peaks were summed. Gentamicin C2 and C are isomers that this assay was unable to resolve; they co-eluted as one peak, so the chromatograms showed 3 rather than 4 peaks. Other authors have quantified gentamicin on the basis of the sum of 3 peaks.
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Protocol for Stability Study
Three formulations were evaluated for compatibility and stability. These were prepared, using aseptic technique, from the commercially available gentamicin (40 mg/mL) and trisodium citrate (46.7% for injection, diluted with water). Formulation A consisted of gentamicin 2.5 mg/mL, formulation B consisted of sodium citrate 40 mg/mL, and formulation C consisted of both gentamicin and sodium citrate at concentrations of 2.5 and 40 mg/mL, respectively. A 30-mL volume of each formulation was prepared, and 5-mL aliquots of each formulation were packaged into six 5-mL polyethylene syringes. The syringes were closed with luer-lock syringe tips and were stored at room temperature in a plastic bin with an opaque, snug-fitting lid.
On days 0, 7, 14, 28, 42, 56, and 112, a sample of about 0.8 mL was withdrawn from each of 4 randomly selected syringes for each formulation. These samples were placed in 1.8-mL screw-top cryovials and stored at —20°C until analysis. Both gentamicin and citrate have been shown to be stable for 6 months when frozen and stored at -20°C. For gentamicin analysis, the samples were diluted 1:1 with water to give a nominal concentration of 250 g/mL; phenylisocyanate derivatives were prepared from 0.5-mL volumes of these solutions, as previously described, and analyzed by means of HPLC. For citrate analysis, samples were diluted 1:25 with water to generate a nominal concentration of 1600 |ig/mL, and these solutions were analyzed by HPLC. The concentration of gentamicin and citrate was quantified with reference to standards analyzed concurrently with the samples. At the time of analysis, all samples, standards, and a blank were coded and analyzed in random order.
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Validation of Gentamicin Assay
Three solutions of gentamicin standard (40 mg dissolved and made up to 100 mL in water) were prepared. These solutions were further diluted with water to prepare 3 sets of gentamicin solutions with concentrations of 80, 160, 240, 320, and 400 ^g/mL. Phenylisocyanate derivatives were prepared from samples of each of these solutions, and the resulting solutions were analyzed as described above. These data were used to calculate the linearity and range of the method, with linear regression of response or peak area as a function of concentration.
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Chemicals, Reagents, and Materials
The study was performed between November 2007 and May 2008. Commercially prepared gentamicin 40 mg/mL for injection (Sandoz Canada Inc, Boucherville, Quebec; lot 139819, expiry May 2009), sodium citrate 46.7% for injection (TriCitrasol, Cytasol Laboratories Inc, Braintree, Massachusetts; lot B82T, expiry January 2011), and USP-grade sterile water for injection (Hospira Health Care Corp, Montreal, Quebec; lot 74-337-DK, expiry January 2011) were used to prepare the study solutions, which were stored in polyethylene syringes with luer-lock caps (MedXL Inc, Montreal, Quebec; lot F86331). Gentamicin sulphate USP (PCCA Canada, London, Ontario; lot C104339) and sodium citrate USP (Spectrum, Gardenia, California; lot KC066) were used as reference standards. High-performance liquid chromatography (HPLC)-grade acetonitrile (lot 891499) and phosphoric acid (lot 082037) and analytical-grade hydrochloric acid (lot 4100030) and sodium hydroxide (lot 016045) were purchased from Fisher Scientific (Nepean, Ontario). Hydrogen peroxide USP (6%) was purchased from Pure Standard Products (Edmonton, Alberta; lot 189). Phenylisocyanate (Fluka; lot 1378398), triethylamine (lot 047K1041), and trifluoroacetic acid (Fluka; lot 046710) were purchased from Sigma-Aldrich (St Louis, Missouri). Samples for analysis were stored frozen in cryovials (Nunc, Rochester, New York; lot 089675), and the mobile phase was filtered through Supor-200 polyethersulfone 0.2-^m membranes (Pall, Gelman Laboratories, East Hills, New York; lot 51321).
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INTRODUCTION
Catheter-related infections are a major problem for hemodialysis patients with central venous catheters for vascular access. Bacteremia may result from microorganisms entering the bloodstream at the catheter insertion site or from contamination of the catheter lumen. Various strategies may be used to prevent this complication, including aseptic precautions when manipulating or accessing the catheter and scrupulous hygiene of the skin around the catheter exit site.
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