Simultaneous Determination of Anti-Infective Drugs Using HPLC–UV

Researchers from the University of Regensburg in Regensburg, Germany developed a new liquid chromatography (LC)-based method for determining ceftazidime and avibactam, two types of anti-infective drugs. Their findings were published in the Journal of Chromatography Open (1).

Ceftazidime scientific molecular model, 3D rendering | Image Credit: © Alexey Novikov - stock.adobe.com

Ceftazidime is a semisynthetic third-generation cephalosporin, which is used to treat bacterial infections in different parts of the body (2). Introduced into clinical practice in the 1980s, it works by killing bacteria or preventing further growth; however, this medicine will not work for colds, flu, or other viral infections. Using third-generation cephalosporins has become compromised by the increasing variety of beta-lactamases within Gram-negative bacilli. Avibactam is a potent inhibitor of many beta-lactamases, which protect substrate drugs from hydrolysis by Gram-negative organisms. It also acts as an antibiotic medication for treating bacterial infections in the body with ceftazidime (3). Avibactam can treat intra-abdominal infections, complicated urinary tract infections, and more.

Anti-infective drugs are typically evaluated pharmacokinetic/pharmacodynamic (PK/PD) indices, which should be expressed as functions of pharmacologically active concentrations. As of the time of publication, only gradient liquid chromatography–tandem mass spectrometry (LC–MS/MS) had been described for simultaneously determination of ceftazidime and avibatam in humans.

In this study, a new high-performance liquid chromatography–ultraviolet (HPLC–UV) method is described for simultaneously determining total or free ceftazidime and avibactam in serum, which is suitable for therapeutic drug monitoring (TDM) or pharmacokinetic studies in humans. Sufficient retention of avibactam, which is polar, was reached by using tetrabutylammonium hydrogen sulfate (TBA) as an ion pairing agent. The sample treatment for determining total serum concentrations followed a repeatedly published protocol, albeit with minor modifications. Namely, serum (100 µL) was mixed with 20 mM sodium phosphate buffer (pH 6.0, 200 µL) and acetonitrile (500 µL). After separation of the precipitated proteins and extraction of acetonitrile with dichloromethane (1.3 mL), an aliquot (1 µL) of the aqueous layer was injected into the HPLC system.

The HPLC method was validated following the ICH guideline M10 on bioanalytical method validation and study sample analysis (4). The lower limit of detection (LOD) was defined as the amount of analyte injected into the column, with a signal-to-noise (S/N) ratio of 3:1. Stability of ceftazidime and avibactam was analyzed in serum of healthy volunteers, in patient serum, in the stock solutions (10 mM sodium phosphate buffer, pH 6), and in 0.9% saline at different temperatures (-70 °C, -20 °C, 6 °C, and at 20–22 °C room temperature) over various time periods as indicated. Furthermore, autosampler stability of the processed samples (6 °C, 20–24 h) was assessed.

The scientists found that the present HPLC–UV method could simultaneously determine ceftazidime and avibactam in patients following therapeutic doses of ceftazidime and avibactam. Sufficient retention of avibactam, which is very polar, was achieved by adding tetrabutylammonium hydrogen sulfate as ion pairing agent. This allowed the determination of both drugs in plasma via isocratic elution. Ceftazidime and avibactam eluted after 3.3 min and 4.8 min, respectively. Further, the assay’s sensitivity was sufficient to describe the plasma pharmacokinetics of ceftazidime and avibactam in patients following intravenous infusion of ceftazidime/avibactam 2.0/0.5 g.

References

(1) Lier, C.; Kees, F.; Witowski, A.; Rahmel, T.; Pockes, S.; Dorn, C. Simultaneous Determination of Ceftazidime and Avibactam in Patients by Isocratic Ion-Pair Liquid Chromatography with Photometric Detection. J. Chromatogr. Open 2025, 7, 100212. DOI: 10.1016/j.jcoa.2025.100212

(2) Ceftazidime (Injection Route). Mayo Clinic 2025. https://www.mayoclinic.org/drugs-supplements/ceftazidime-injection-route/description/drg-20073334 (accessed 2025-4-22)

(3) Avibactam. Drugbank 2025. https://go.drugbank.com/drugs/DB09060 (accessed 2025-4-22)

(4) ICH M10 on Bioanalytical Method Validation – Scientific Guideline. European Medicines Agency 2022. https://www.ema.europa.eu/en/ich-m10-bioanalytical-method-validation-scientific-guideline (accessed 2025-4-22)