HPLC Method for Quantification of Caffeine and Its Three Major Metabolites in Human Plasma Using Fetal Bovine Serum Matrix to Evaluate Prenatal Drug Exposure

Abstract

Caffeine is recognized as the first-line therapeutic agent for apnea of prematurity. The dosage regimen is 10 mg/kg loading dose and 2.5 mg/kg maintenance dose. However, the plasma concentration achieved, not always, is therapeutically useful. It makes necessary to increase the doses to reach plasma concentration up to 30 or 35 μg/mL or even higher to attain therapeutic effect. To study why neonates have these differences, and whether these effects are linked to prenatal caffeine exposure, we had to develop an analytical method for an accurate measurement of caffeine and metabolites concentration. The analysis was carried out using fetal bovine serum (FBS) as biological matrix in a high-performance liquid chromatography with an ultraviolet detector method. This method allows acceptable chromatographic resolution between analytes in 15 minutes. It was validated and proved to be linear in the 0.1–40 µg/mL range for caffeine, paraxanthine, theobromine, and theophylline in the same chromatographic analysis. Accuracy for quality control samples for intra- and interday assays was ranged from 96.5 to 105.2% and 97.1 to 106.2%. Precision had CV no more than 10% in all concentration levels for all analytes. No differences were observed between quantification in human and FBS. This method was applied to quantify plasma drug concentration in mothers and their newborns in a Mexican northeast population. In our study, we confirmed self-reported caffeine maternal intake in 85.2% (n=23); meanwhile, in their newborn's plasma, it was detected only in 78% (n=21). Caffeine plasma concentrations in mother and newborn had a linear relationship, and no differences were observed between groups (mothers versus children). These results suggest that our analytical method and substitution of biological matrix was linear, precise, and accurate for caffeine quantification and could be used for measuring prenatal exposure and let us to study, in the future, concentration differences observed during apnea clinical treatment.

Validation of an LC-MS/MS Method for the Quantification of Caffeine and Theobromine Using Non-Matched Matrix Calibration Curve

“…There are several methods described in the literature for the simultaneous absolute quantification of caffeine and their main three metabolites in plasma [18,33,34,35]. The sample preparation is generally very similar involving a protein precipitation step using an organic solvent (methanol) and in some cases adding a filtration or solid-phase extraction (SPE) step before the LC-MS analysis.…”

“…Although caffeine and its metabolites are exogenous compounds, the same challenge of obtaining human plasma without the compounds of interest occurs during the method validation process. There are some matrices already validated for caffeine quantification which were used to prepare synthetic plasma without caffeine [39], and fetal bovine serum matrix [34]. However, none of these matrices truly represents the original matrix and finding caffeine-free human plasma volunteers can be challenging.…”

Caffeine inhibits Notum activity by binding at the catalytic pocket

“…It is therefore unlikely that moderate coffee drinking would result in physiological Notum inhibition. However, caffeine is used as a therapeutic agent at much higher concentrations, for example in the treatment of apnea of prematurity where the plasma concentration can reach 150 µM 37 . At this concentration caffeine does not appear to have adverse effects on the brain 38 but would likely cause Notum inhibition.…”

Liquid Chromatography Analysis of Common Nutritional Components, in Feed and Food

“…Caffeine is a demonstrated therapeutic agent for apnea of prematurity. Hence, López-Sánchez developed a method to monitor caffeine in serum to demonstrate that the drug had achieved its therapeutic levels (i.e., 30 or 35 μg mL −1 ) [161]. Cleanup using SPE adapted in multiple well plates, as the one used in the former study, is an easy way to process several samples simultaneously, instead of the one-on-one cartridge approach.…”