4. A LC-MS assay was developed and validated in the laboratory to quantify penicillin G (antibiotic) from human blood serum.
Serum samples were thawed at room temperature and 50 µL of serum was transferred into a 0.2 mL tube. For a serum an extraction 50 µL of acetonitrile (containing piperacillin as internal standard (I.S.) in a concentration of 10 µg/mL) was added. After vigorous shaking in a Vortex mixer for 2 min, the sample was centrifuged at 13 000 rpm for five minutes until the supernatant (approximately 75 µL) was separated. The supernatant was then filtrated through a 0.22 µm filter and transferred into ultra high performance liquid chromatography (UHPLC) autosampler vials using inserts.
Samples were analysed using UHPLC coupled with triple quadrupole. ESI source was used in the positive ionization mode.
When study samples arrived to the laboratory for the analysis, then approximately 20% of them were visually haemolysed (serum contains red blood cells) and 10% hyperlipidemic (serum contains abnormal amount of lipids).
Please evaluate the possible solutions to test matrix robustness during the validation.
| Possible solution |
Evaluation |
| Blood cell content does not affect the assay performance and can be left out during the matrix robustness/selectivity studies |
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| Lipid concentration has low effect on the assay performance and can be left out during the matrix robustness/selectivity studies |
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| Use different blood serums from 6 different sources to evaluate the matrix robustness. |
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| Evaluate the effect of haemolysis with spiking the blood serum with different amount (in the range of 0-10%) of whole blood and evaluate the effect of blood content for the assay performance |
|
| Use matrix with ≥ 240 mg/dL (total) cholesterol available from a commercial source to evaluate the effect of hyperlipidemic blood serum |
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