A Validated LC-MS/MS Method for the Quantitation of Cefazolin in Human Adipose Tissue: Application of EMR-Lipid Sorbent As an Efficient Sample Clean-Up Before Mass Spectrometric Analyses.

A novel, simple, rapid, and sensitive high-performance liquid chromatography-tandem mass spectrometry method was developed to determine cefazolin concentrations in human adipose tissue. Sample preparation was performed by protein precipitation followed by using Captiva EMR-Lipid plates. The mobile phase consisted of 5 mM ammonium formate and 0.1% formic acid in water and 0.1% formic acid in ACN, and was pumped through a Synergi Fusion-RP column with a gradient elution program at a flow rate of 0.3 mL/min. The mass spectrometer was operated in a positive ion mode. Cloxacillin was used as an internal standard due to the observed cross-signal contribution between cefazolin and C-13(2), N-15-cefazolin. The method was validated according to the FDA and EMA guidelines and passed all the acceptance criteria. The calibration range was 0.05-50 mu g/mL in adipose tissue homogenate (0.15-150 mu g/g in adipose tissue), precision CV < 4.5%, accuracy within 93.1-100.4%. The carryover was negligible, recovery of the method was high, and no significant matrix effect was present. Rat subcutaneous adipose tissue was demonstrated to be a suitable surrogate matrix for human adipose tissue. The validated method was successfully applied in a pilot pharmacokinetic study and will further be used in a large cohort of non-obese and obese patients dosed prophylactically with cefazolin before surgeries.