Self test 10.3

2. A LC-MS assay was developed and validated in the laboratory to measure meropenem (antibiotic; pK_a1=3.47; pK_a2=9.39) from human blood serum.

Blood serum samples were thawed at room temperature and 50 µL of serum was transferred into 250 µL tube. For serum sample extraction 50 µL of methanol (containing ertapenem (pK_a1=3.37; pK_a2=9.03) as internal standard (I.S.) in a concentration of 10 µg/mL) was added. After vigorous shaking with Vortex mixer for 1 min, the sample was centrifuged at 8 000 rpm (3500 × g) for 10 min and supernatant (approximately 75 µL) was separated and filtrated through 0.22 µm filter and transferred into autosampler vial.

Samples were analysed using UHPLC coupled with triple quadrupole. Gradient elution with methanol and 0.1 % formic acid (pH = 2.6) on a flow rate of 0.3 mL/min was used for chromatographic separation. Samples were chromatographed using C18 column (2.1 × 100 mm, 1.7 µm) equipped with C18 pre-column (2.1 × 5 mm, 1.7 µm). Column temperature was set to 40 ⁰C. ESI source (allows the change in source configuration) was used in the positive ionization mode. Transitions of the parent ion with m/z 384 [M + H]⁺ to daughter ions m/z 254, 298, 340 were used for meropenem quantification and qualification.

The calibration curves were linear from 0.1 to 200 µg/mL in serum. LoQ for serum samples was 0.1 µg/mL.

Please evaluate the effect (high, medium, low) of different method parameters that are affecting method robustness and what can actually change during the analysis of study samples.