Accurate analysis of antibiotics performed with dry blood spots

Measuring antibiotic levels in patients with pneumonia is a challenge

Hospital-acquired pneumonia is prevalent in many hospitals, usually affecting approximately 1% of hospitalized patients. It kills more patients than any other infection acquired in the hospital, and the fans are particularly at risk. Treatment is usually through a combination of antibiotics, such as linezolid and ciprofloxacin. It is important to be able to control the concentration of antibiotics in the blood of patients; Larger hospitals often have a TDM (therapeutic drug monitoring) laboratory to do this, but in smaller hospitals these are nonexistent or can be quite rudimentary. Conventional monitoring methods, which typically require long extraction techniques followed by HPLC, are not practical in such units. Instead, simple techniques are required that also provide quick and accurate results.

Dry blood spots (DBS) are a quick and direct way to test therapeutic drug levels in patients. In this technique, a blood capillary sample of a puncture is placed on a filter paper or on a DBS card for drying. The HPLC can be carried out on the dried sample, if necessary after transport elsewhere. Italian scientists examined the use of DBS to monitor the levels of linezolid and ciprofloxacin, with the aim of developing an accurate method suitable for widespread use.
DBS and HPLC used to monitor antibiotic levels

Blood from the volunteers was increased with linezolid, ciprofloxacin and an internal standard (the antibiotic ulifloxacin). Pricking samples were also taken from patients treated with linezolid and ciprofloxacin, with venous blood samples taken for comparison. Both tipped blood samples and finger prick samples were plated on Bond Bond DMS plates, allowed to dry and then extracted with 80% aqueous methanol containing 0.1% formic acid under sonication. The extraction conditions were carefully optimized to give a good recovery. The solids were removed by centrifugation, the solvent was 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% triethylamine v / a was added both A and B in order to obtain a better peak shape). The gradient ranged between 90 and 65% of solution A at 1.0 ml / min. Detection was at 251 nm for linezolid and 277 nm for ciprofloxacin. HPLC gave a good separation of linezolid, ciprofloxacin and the internal standard.

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

It was found that the general method provided 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. The results of finger punctures (capillary blood) were in line with those of the venous blood. The results of DBS correlated well with conventional plasma assays.
DBS is a convenient technique for testing compounds in blood

DBS has proven to be an effective and simple technique for the testing of antibiotics in the blood. The new technique allows rapid tests with only basic laboratory equipment (simple glassware, centrifuge, standard HPLC with UV detector). Blood spots are also easier to transport than whole blood samples, saving time and cost. 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 document.