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Enzyme immunoassay of gentamicin with use of a centrifugal analyzer
Abstract:
Gentamicin was determined in serum by the homogeneous enzyme immunoassay system (EMIT) adapted to the Multistat centrifugal analyzer. The standard curve can be extended to 16 mg/L; however, poor precision is obtained at concentrations greater than 10 mg/L because a small difference in change in absorbance there will produce a significant variation in gentamicin concentration. For example, the within-run precision of the method (CV) was 3.4% for a 5.0 mg/L sample (range 4.6--5.4 mg/L) and 11.1% for a 12.2 mg/L sample (range 10.4--15.4 mg/L). We recommend that all samples containing gentamicin at concentrations exceeding 10 mg/L be diluted with Tris buffer. Recovery of gentamicin added to four serum specimens averaged 102%. There is no interference from bilirubin at concentrations up to 200 mg/L or from moderately lipemic samples. Hemoglobin in serum in excess of 625 mg/L results in lower gentamicin values. Gentamicin is stable for six days in serum stored at 23, 4, or -20 degrees C. Comparison of gentamicin results by the proposed method with those by a manual EMIT procedure, and with those by a homogeneous fluorescence immunoassay method gave correlation coefficients of 0.986 and 0.947.
Insights
This study validates a homogeneous enzyme immunoassay (EMIT) for gentamicin in serum using a centrifugal analyzer. Dilution is recommended for samples above 10 mg/L to maintain assay precision and accuracy.
Area of Science:
- Clinical Chemistry
- Pharmacokinetics
- Analytical Toxicology
Background:
- Gentamicin is a crucial antibiotic requiring therapeutic drug monitoring.
- Accurate quantification of gentamicin in serum is vital for patient safety and efficacy.
- Existing immunoassay methods may have limitations in precision at higher concentrations.
Purpose of the Study:
- To adapt and validate a homogeneous enzyme immunoassay (EMIT) for gentamicin determination in serum.
- To assess the performance characteristics of the EMIT method on a Multistat centrifugal analyzer.
- To establish optimal conditions for accurate gentamicin measurement in clinical samples.
Main Methods:
- Homogeneous enzyme immunoassay (EMIT) adapted for automated analysis.
- Utilized a Multistat centrifugal analyzer for sample processing.
- Evaluated standard curve linearity, precision, recovery, and interference from common serum components.
Main Results:
- The EMIT method demonstrated good precision (CV 3.4%) at 5.0 mg/L, but reduced precision (CV 11.1%) above 10 mg/L.
- Recommended dilution with Tris buffer for samples exceeding 10 mg/L.
- Average recovery was 102%, with no significant interference from bilirubin or moderate lipemia.
- Hemoglobin above 625 mg/L caused lower gentamicin values.
- Gentamicin stability in serum was confirmed for up to six days across various temperatures.
- High correlation coefficients (0.986 and 0.947) were observed when compared to manual EMIT and fluorescence immunoassay methods.
Conclusions:
- The adapted EMIT method provides a reliable and automated approach for gentamicin monitoring in serum.
- Dilution of high-concentration samples is crucial for maintaining assay precision.
- The method shows good recovery and minimal interference, supporting its clinical utility.