Bristol Centre for Antimicrobial Research and Evaluation, Department of Microbiology, Southmead Hospital, Bristol BS10 5NB, UK
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Abstract |
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Introduction |
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With all new drug therapies it is important to have an assay to individualize therapy, for pharmacokinetic studies and to investigate any possible drug interactions. The aim of this study was to develop such an assay.
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Materials and methods |
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These were as follows: amikacin (Bristol-Myers Squibb, Syracuse, NY, USA); amoxicillin, ampicillin, azlocillin, cefotaxime, cefuroxime, cycloserine, chloramphenicol, gentamicin, norfloxacin, rifampicin, sulfamethoxazole and tobramycin (Sigma, Steinheim, Germany); ceftazidime and aciclovir (GlaxoSmithKline, Greenford, UK); ciprofloxacin and moxifloxacin (Bayer AG, Wuppertal, Germany); flucloxacillin (GlaxoSmithKline, Welwyn Garden City, UK); 5-flucytosine and ganciclovir (Roche, Welwyn Garden City, UK); linezolid (Pharmacia, Kalamazoo, MI, USA); meropenem (AstraZeneca, Södertälje, Sweden); netilmicin (Schering-Plough, Kenilworth, NJ, USA); ofloxacin (Aventis Pharma, West Malling, UK); penicillin-G (Britannia, Redhill, UK); and vancomycin (Eli-Lilly, Indianapolis, IN, USA).
Solvents
Acetonitrile, methanol and water (Chromanorm grade) were purchased from Prolabs (Fontenay, France); 1-heptane sulphonic acid was supplied by Sigma; ortho- phosphoric acid and sodium hydroxide by BDH (Analar grade; Poole, UK).
High-performance liquid chromatography (HPLC)
The stationary phase was Hypersil 5ODS, 10 cm x 4.6 mm (Waters Corporation, Milford, MA, USA).
The mobile phase was 1% ortho-phosphoric acid, 30% methanol, 2 g/L heptane sulphonic acid, adjusted to pH 5 by the addition of 10 M sodium hydroxide. The pump flow rate was 1.0 mL/min. UV absorbance detection was used (max 254 nm). A Gina 50 autosampler was used (Dionex, Macclesfield, UK) and the integrator was a Trilab 2000 (Trivector, Sandy, UK).
Sample preparation
Samples were prepared by mixing aliquots (50:50) of the specimen with acetonitrile. The samples were mixed, allowed to rest at ambient temperature for 10 min and centrifuged at 5000g for 5 min. Twenty microlitres of the supernatant was injected.
Reproducibility
Serum specimens containing 5, 15 and 30 mg/L linezolid were assayed (n = 3). Mean peak heights ± s.d. (x ± s.d.) and percentage coefficients of variation (% CV) were calculated [(s.d./mean) x 100] over a single day to assess intra-day reproducibility and on days 1, 2, 6 and 7 of the evaluation to assess inter-day reproducibility.
Linezolid stability
All stability determinations (apart from the freezethaw investigations) were carried out using 0.4, 25 and 45 mg/L linezolid serum samples. Initially, the drug stability was assessed over a 24 h period after acetonitrile sample preparation. Linezolid stability after storage at 4°C and at room temperature was determined by assay (n = 3) on day 7 of the study. For these stability investigations, the calculated concentration on day 1 was compared with that measured on day 7, and the percentage original calculated [(day 2 or 7 concentration/day 1 concentration) x 100]. Linezolid was also subjected to two heat treatment cycles (56°C for 45 min; serum samples from high-risk groups are virally inactivated in this way). Drug stability was determined by assay (n = 6) of samples before heat treatment and re-assay (n = 3) after the first cycle. After cooling to room temperature, the samples were subjected to a further cycle and the sample assayed again (n = 3). Calculated concentrations before heat treatment were compared with those after each cycle and the percentage of the original concentration calculated. Linezolid stability was assessed after two freezethaw cycles. This was determined by assay (n = 3) of a serum linezolid 8 mg/L sample thawed after storage at 70°C. The sample was returned to the freezer, frozen, thawed and assayed (n = 3) once more. The percentage weighed-in concentration was calculated after each cycle. The long-term stability of linezolid was assessed by the assay (n = 3) of 0.4, 25 and 50 mg/L linezolid serum samples after storage at 70°C for 1 year. The measured concentrations were compared with those of the weighed-in samples.
Linearity
Linearity was determined with serum samples spiked with 0.0, 0.5, 1, 5, 10, 15, 20 and 30 mg/L linezolid. The samples were assayed (n = 3) and the mean peak height was calculated and plotted against sample concentration. Linearity was investigated via regression analysis.
Recovery
This was investigated with serum and aqueous samples spiked with 0.0, 0.5, 1, 5, 10, 15, 25 and 30 mg/L linezolid. The samples were assayed (n = 6) and peak heights measured. The mean peak height was plotted against linezolid concentration and the percentage recovery calculated [(mean aqueous peak height/mean serum peak height) x 100].
Lowest limit of quantification
The lowest limit of quantification is defined as the concentration equivalent of a peak approximately three times the height of the baseline noise. A linezolid serum sample was prepared to approximately this concentration. The sample was assayed (n = 3) and peak height ± s.d. and % CV calculated.
Specificity
The assay was evaluated for any possible interference from other antimicrobial, antifungal and antiviral drugs that may be co-administered with linezolid. The antimicrobial agents for assessment were prepared to contain clinically appropriate concentrations. After sample preparation, the following were assayed: aciclovir 20 mg/L, amikacin 53 mg/L, ampicillin 20 mg/L, amoxicillin 26 mg/L, azlocillin 58mg/L, benzyl-penicillin 20 mg/L, cefotaxime 46 mg/L, cefuroxime 14 mg/L, chloramphenicol 19.4 mg/L, ciprofloxacin 2.6 mg/L, cycloserine 26.2 mg/L, flucloxacillin 74 mg/L, 5-flucytosine 67 mg/L, ganciclovir 20 mg/L, gentamicin 5.7 mg/L, meropenem 15 mg/L, moxifloxacin 5 mg/L, netilmicin 0.7 mg/L, norfloxacin 5 mg/L, ofloxacin 5 mg/L, rifampicin 5 mg/L, sulfamethoxazole 115 mg/L, tobramycin 9.8 mg/L, trimethoprim 8 mg/L and vancomycin 59.5 mg/L.
The method was also assessed for any interference from unknown endogenous compounds in patient sera. Twelve linezolid-free serum samples (sent to our laboratory for antibiotic assay) were selected at random. The samples were prepared and assayed.
Accuracy using single point calibration
The accuracy of the method was evaluated by the preparation and assay (n = 6) of serum samples spiked to contain 2.5, 8 and 18 mg/L linezolid using a 12 mg/L serum calibrator.
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Results |
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The separated linezolid peak is shown in Figure 1. Assay reproducibility was: intra-day <6%; inter-day <12.5% (Table
). The correlation between drug concentration and peak height was good for both aqueous and serum samples across the concentration range (r = 0.9999 for both; Figure 2
). Linezolid recovery from serum approached 100% at all concentrations tested. The lowest limit of quantification was calculated to be 0.1 mg/L. The accuracy, expressed as the percentage error [100 x (measured concentration target concentration)/target concentration] was 4.0 for the 2.5 mg/L sample, 1.3 for the 8 mg/L sample and 0.0 for the 18 mg/L sample. There was no interference from 23 commonly used antimicrobial agents or from any unknown compound present in linezolid-free patient sera. Benzyl-penicillin eluted close to linezolid. However, this was not considered to be significant since benzyl-penicillin 20 mg/L resulted in a peak equivalent to linezolid 0.4 mg/L. Furthermore, benzyl-penicillin can be removed by ß-lactamase.
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Linezolid was stable in serum, after sample preparation with acetonitrile, at room temperature over a 24 h period (Figure 3). The drug was stable in serum alone for at least 7 days at both room temperature and 4°C.
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Discussion |
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The assay is reproducible, accurate and linear across the concentration range. Linezolid was fully recoverable from serum. The lowest limit of detection (0.1 mg/L) is somewhat lower than the MICs for control organisms (0.54 mg/L; Linezolid Technical Information, unpublished). The only published HPLC method for linezolid has been validated for dog, mouse, rabbit and rat plasma.8 This method uses a time-consuming and complicated sample preparation step using solid-phase extraction. With our method, sample preparation is quick and simple, and up to 150 assays can be performed before there is a change in chromatographic performance. With a retention time of only 6.5 min, it is ideal for the processing of clinical samples.
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Acknowledgements |
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Notes |
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References |
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8 . Peng, G. W., Stryd, R. P., Murata, S., Igarashi, M., Chiba, K., Aoyama, H. et al. (1999). Determination of linezolid in plasma by reversed-phase high-performance liquid chromatography. Journal of Pharmaceutical and Biomedical Analysis 20, 6573.[ISI][Medline]
Received 28 March 2001; returned 14 June 2001; revised 12 July 2001; accepted 8 August 2001