1 Department of Clinical Biochemistry, Wythenshawe Hospital, Southmoor Road, Manchester M23 9LT; 2 Department of Medical Microbiology, Southmead Health Services NHS Trust, Bristol BS10 5NB, UK
Received 20 September 2001; returned 13 February 2002; revised 7 March 2002; accepted 25 March 2002
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Abstract |
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Introduction |
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The Association of the British Pharmaceutical Industry (ABPI) data sheet recommends that in serious infections steady-state trough teicoplanin concentrations be maintained above 10 mg/L. In addition, it recommended that a minimal trough concentration of 20 mg/L be maintained for optimal monotherapy of S. aureus endocarditis. Standard dosing does not reliably produce trough concentrations greater than 10 mg/L in seriously ill patients and those with renal impairment.3 Monitoring is therefore recommended and should be practised on selected patients.
Measurement of teicoplanin can be carried out using HPLC methods of varying complexity, microbiological assays, which are labour intensive and time consuming, and more commonly immunoassay.4 There is, however, only one immunoassay on the market (TDx FPIA; where FPIA stands for fluorescence polarization immunassay), which may not be available indefinitely and may not be available worldwide. For this reason it is important that a clinically validated and simple HPLC method should exist as a possible replacement for teicoplanin measurement.
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Materials and methods |
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Equipment and materials
The chromatographic apparatus consisted of a Thermo Separation Products (TSP, Hemel Hempstead, UK) P100 pump, TSP AS300 autosampler and TSP UV100 spectrophotometer. The data were acquired and processed using TSP PC1000 software.
The column used was a 150 x 4.6 mm, 5 µm particle size, C8 Sphereclone column (Phenomenex, Macclesfield, UK). Chloroform, acetonitrile (both HPLC grade), ammonium acetate and glacial acetic acid were purchased from BDH Chemicals Ltd (Poole, UK). Teicoplanin sodium salt (batch number 0248\2) was obtained from Hoechst Marion Roussel (Uxbridge, Middlesex, UK).
Mobile phase
The mobile phase consisted of acetonitrile/30 mM ammonium acetate (26:74). The ammonium acetate buffer was adjusted to pH 4.4 with glacial acetic acid before the addition of acetonitrile. The solution was de-gassed by filtration through 0.2 µm pore nylon Rainin-66 filters. The chromatographic separations were performed isocratically at a flow rate of 2.5 mL/min. UV detection was at 218 nm, sensitivity range 0.002, absorption units full scale and rise time 1.0 s.
Preparation of samples and calibrators
Forty-one serum samples obtained over a 1 month period from patients on teicoplanin, and originally assayed by FPIA and stored frozen at 20°C were thawed and re-analysed by the HPLC method.
A 5000 mg/L teicoplanin serum-based superstock was made by adding 50 mg of teicoplanin to 10 mL of pooled serum. A 1 in 10 dilution of the superstock in pooled serum was used to make a 500 mg/L stock solution. A 1 in 5 dilution of the stock teicoplanin in pooled serum was used to make a 100 mg/L calibrator and a 1 in 10 dilution of this in pooled serum was used to make the 10 mg/L calibrator. Aliquots of these calibrators were dispensed and stored at 20°C.
Sample preparation consisted of adding 400 µL of acetonitrile to 200 µL of sample, thawed calibrator or internal control sample and vortexing for 20 s. After centrifuging for 2 min at 2000g using an Abbott benchtop microcentrifuge the supernatant was removed leaving the protein pellet behind. Chloroform (800 µL) was added to the first supernatant, vortexed for 20 s and centrifuged as before. The final supernatant was removed and used for the analysis of teicoplanin, injecting 50 µL on to the column.
FPIA
Teicoplanin was assayed by FPIA. The reagents were obtained from Bio-Stat Diagnostis Ltd (Stockport, UK), and the tests were carried out on the TDx analyser (Abbott Laboratories, Chicago, IL, USA). This method, reviewed previously by McMullin et al.6 was shown to have an inter-assay coefficient of variation (S.D./mean x 100) of 5.6% (8.1 mg/L control; n = 8) 1.4% (33.0 mg/L control; n = 8) and 5.8% (74.4 mg/L control; n = 8)
Statistics
Method comparisons were carried out using a paired t-test and the regression line was calculated according to the procedures of Passing & Bablok.7 Agreement was also assessed by the BlandAltman method.8 All statistical analysis was carried out on the Analyse-it software package (University of Leeds, UK).
Recovery experiment
Patient samples were spiked with the serum-based 100 mg/L calibrator and the re-measured teicoplanin levels compared with the theoretical values to give a measurement of recovery.
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Results and discussion |
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The limit of detection calculated as a magnitude of 2.5 times the signal noise in a blank sample was 0.05 mg/L. The functional sensitivity of the assay was not determined; however, the inter-assay coefficient of variation at 10 mg/L was 2.76%, which demonstrates adequate precision at the lowest clinically relevant cut-off level.
The FPIA and HPLC regression was good: HPLC = 0.908 TDx + 2.324 (see Figure 2a) with r2 = 0.974. There was no concentration-dependent bias between the results as shown in the BlandAltman difference plot (Figure 2b), and the differences between paired samples were normally distributed. A paired t-test showed that the results were not significantly different (P = 0.6531). We originally constructed a standard curve using 10, 50, 75 and 100 mg/L calibrants; however, due to good linearity on the curve (peak height = 2030 teico + 11470, r2 = 0.9996), only two standards, 10 and 100 mg/L, were used for future assays including method comparison.
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The reverse-phase isocratic HPLC method described uses very basic apparatus and makes the measurement of teicoplanin accessible to routine laboratories with appropriate equipment. The excellent analytical correlation and overall cost of the HPLC analysis compared with FPIA makes this method a very attractive alternative to FPIA.
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Acknowledgements |
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Footnotes |
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References |
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2 . Somma, S., Gastaldo, L. & Corti, A. (1984). Teicoplanin, a new antibiotic from Actinoplanes teichomyceticus nov. sp. Antimicrobial Agents and Chemotherapy 26, 91723.[ISI][Medline]
3 . Falcoz, C., Ferry, N., Pozet, N., Cuisinaud, G., Zech, P. Y. & Sassard, J. (1987). Pharmacokinetics of teicoplanin in renal failure. Antimicrobial Agents and Chemotherapy 31, 125562.[ISI][Medline]
4 . Awni, W. M., St Peter, W. L., Guay, D. R. P., Kenny, M. T. & Matzke, G. R. (1991). Teicoplanin measurement in patients with renal failure: Comparison of fluorescence polarization immunoassay, microbiological assay and high performance liquid chromatographic assay. Therapeutic Drug Monitoring 13, 5117.[ISI][Medline]
5 . Cociglio, M., Peyriere, H., Hillaire-Buys, D. & Alric, R. (1998). Application of a standard coextractive cleanup procedure to routine high-performance liquid chromatography assays of teicoplanin and ganciclovir in plasma. Journal of Chromatography 705, 7985.
6 . McMullin, C. M., White, L. O., MacGowan, A. P., Holt, A., Lovering, A. M. & Reeves, D. S. (1994). Assay of serum teicoplanin concentrations in clinical specimens: a comparison of isocratic high performance liquid chromatography with polarisation fluorimmunoassay and bioassay. Journal of Antimicrobial Chemotherapy 34, 4259.[ISI][Medline]
7 . Passing, W. and Bablok, H. (1983). A new procedure for testing the equality of measurements from two methods. Journal of Clinical Chemistry and Clinical Biochemistry 21, 70920.[ISI][Medline]
8 . Bland, J. M. & Altman, D. G. (1986). Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1, 30710.[ISI][Medline]