Bioanalytical development and validation of liquid chromatographic-tandem mass spectrometric methods for the quantification of total and free cefazolin in human plasma and cord blood

Abstract

Objectives: Cefazolin is a commonly prescribed β-lactam antibiotic for prophylaxis against skin infections following surgery, including caesarean sections. Assessment of maternal and neonatal exposure is important for correlating drug concentrations to clinical outcomes. Thus, bioanalytical methods for the quantification of both total and free cefazolin in maternal plasma and cord blood can assist in the comprehensive evaluation of cefazolin exposure.

Design and methods: Specimen preparation for the measurement of total cefazolin was performed via protein precipitation with acetonitrile containing the internal standard cloxacillin. Ultrafiltration was used to isolate free cefazolin. Processed samples were analyzed on a Prelude SPLC system coupled to a TSQ triple quadrupole Vantage mass spectrometer. Methods were validated following FDA bioanalytical guidelines.

Results: The analytical measuring ranges of these methods were 0.48-480 µg/mL and 0.048-48 µg/mL for total and free drug, respectively. Calibration curves were generated using 1/x² weighted linear regression analysis. Total cefazolin demonstrated inter- and intra-assay precision of ≤20% at the LLOQ and ≤11.2% at other levels. Free cefazolin demonstrated inter- and intra-assay precision of ≤18.5% at the LLOQ and ≤12.6% at other levels, respectively. Accuracy (%DEV), carryover, matrix effects, recovery and stability studies were also acceptable based on FDA recommendations. Furthermore, it was demonstrated that samples prepared in cord blood can be accurately quantified from an adult plasma calibration curve, with recoveries ≤9.1% DIF and ≤11.9% DIF for total and free cefazolin, respectively.

Conclusions: The described LC-MS/MS methods allow for the measurement of total and free cefazolin in both plasma and cord blood.

Keywords: Antibiotic; Cefazolin; Mass spectrometry; Method validation; Ultrafiltration.

Fig. 1

Structures of (A) the beta-lactam antibiotic cefazolin and its (B) structural analog, cloxacillin.

Fig. 2

Representative chromatograms for (A) cefazolin LLOQ, low QC and blank plasma and (B) cloxacillin internal standard and blank plasma.

Fig. 3

Representative chromatograms of carryover optimization sequences. Chromatograms from (A) are prior to optimization. Chromatograms from (B) are post-optimization.

Fig. 4

Representative calibration curves for total cefazolin analysis. The y-axis of (A) is the peak area ratio observed between cefazolin and cloxacillin quantifier transitions. The y-axis of (B) is the calculated concentration using linear regression.