Accurate antibiotic analysis achieved with dried blood spots

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  • Published: May 15, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Accurate antibiotic analysis achieved with dried blood spots

The measurement of antibiotic levels in pneumonia patients is a challenge

Hospital-acquired pneumonia is prevalent in many hospitals, typically affecting approximately 1% of in-patients. It kills more patients than any other hospital-acquired infection, with those on ventilators being particularly at risk. Treatment is usually by means of a combination of antibiotics, such as linezolid and ciprofloxacin. It is important to be able to monitor the concentration of antibiotics in patients’ blood; larger hospitals typically have a TDM (therapeutic drug monitoring) laboratory to do this, but in smaller hospitals these are either non-existent or may be quite rudimentary. Conventional monitoring methods, which typically require lengthy extraction techniques followed by HPLC, are not practical in such units. Instead, simple techniques that also give speedy and accurate results are required.

Dried blood spots (DBS) are a quick and straightforward way to test for the levels of therapeutic drugs in patients. In this technique, a capillary blood sample from a finger-prick is placed onto a filter paper or DBS card to dry. HPLC can be carried out on the dried sample, if necessary after transport elsewhere. The Italian scientists examined the use of DBS to monitor levels of linezolid and ciprofloxacin, aiming to develop an accurate method suitable for widespread use.

DBS and HPLC used to monitor antibiotic levels

Blood from volunteers was spiked with linezolid, ciprofloxacin and an internal standard (the antibiotic ulifloxacin). Finger-prick samples were also taken from patients being treated with linezolid and ciprofloxacin, with venous blood samples taken for comparison. Both the spiked blood samples and the finger-prick samples were spotted onto Bond Elut DMS cards, allowed to dry and then extracted with 80% aqueous methanol containing 0.1% formic acid under sonication. The extraction conditions were carefully optimised in order to give good recovery. The solids were removed by centrifugation, the solvent evaporated and the residue was dissolved in mobile phase for HPLC injection.

HPLC was carried out using a Waters 600 pump, a Water 2996 photodiode array detector and a Phenomenex Kinetex EVO C18 column. Gradient elution was carried out with two solutions: solution A was 10 mMol aqueous ammonium acetate, adjusted to pH 3.5 with acetic acid, while solution B was 80/20 v/v acetonitrile/methanol (0.1% v/v triethylamine was added to both A and B in order to obtain a better peak shape). The gradient ran between 90 and 65% of solution A at 1.0 ml/min. Detection was at 251 nm for linezolid and 277 nm for ciprofloxacin. The HPLC gave good separation of linezolid, ciprofloxacin and the internal standard.

When calculating concentrations of antibiotics in the blood spots, it was necessary to correct for the natural variation in haematocrit (Hct) values (this is a measure of the concentration of red blood cells in the blood). Concentrations were corrected using a standard equation, which gave better accuracy and precision than uncorrected results.

The overall method was found to give good precision and linearity. The limits of detection (LOD) were 0.015 μg/ml for linezolid and 0.008 μg/ml for ciprofloxacin, respectively, while the limits of quantification (LOQ) were 0.05 μg/ml for linezolid and 0.02 μg/ml for ciprofloxacin. Results from finger-pricks (capillary blood) were in line with those from venous blood. The DBS results correlated well with conventional plasma assays.

DBS is a convenient technique for assays of compounds in blood

DBS has been shown to be an effective and simple technique for the assay of antibiotics in blood. The new technique allows rapid assays with only basic laboratory equipment (simple glassware, centrifuge, standard HPLC with UV detector). The blood spots are also easier to transport than whole blood samples, again saving on time and costs. It is likely that the combination of DBS and HPLC will be increasingly used in the future, being applicable to many compounds other than the two studied in the paper.

Related Links

Drug Testing and Analysis, 2016, Early View paper. Ferrone et al. Development of a dried blood spot HPLC-PDA method for the analysis of linezolid and ciprofloxacin in hospital-acquired pneumonia patients.

Clinical Pharmacokinetics, 2014, 53, 961-973. Wilhelm et al. Therapeutic drug monitoring by dried blood spot: Progress to date and future directions.

Bioanalysis, 2015, 7:16, 2119-2130. Patteet et al. Are capillary DBS applicable for therapeutic drug monitoring of common antipsychotics? A proof of concept.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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