Development of the High-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Creatine, L-Carnitine, and Creatinine in Tears.

The aim of this work was to develop the HPLC-MS/MS method for the determination of L-carnitine and creatine in tears, as these molecules can be used as biomarkers of several diseases and health conditions with neurological manifestation, simultaneously with the determination of creatinine as normalization factor. Tears were collected using a microtube during crying or using Schirmer strip by inserting it under the lower eyelid. Due to the low stability of creatine, only a few hours at room temperature, it is advisable to freeze the samples immediately after collection. Different methanol to water contents in the extraction solution were tested (0%-100%). Extraction recovery from Schirmer strips, matrix effect, and overall process efficiency were evaluated for 0.3 and 1 mL of extraction solution used. Finally, the extraction into 1 mL of 80% methanol containing 0.1% formic acid, and subsequent HPLC-MS/MS separation at isocratic conditions (methanol:water 5:95% with 0.1% formic acid and flow rate of 0.25 mL/min) using a Synergi 4 μ Polar-RP column (150 × 2 mm) and detection in the positive ion mode was used. The limits of quantifications for L-carnitine, creatine, and creatinine in tears were 0.5, 1.0, and 1.0 µmol/L, respectively. Intraday and interday precisions were up to 4.4% and bias did not exceed 12% for all analytes. Linearity was tested and approved in the range of 0.23-30 µmol/L for L-carnitine and 0.78-100 µmol/L for creatine and creatinine. Measured values of L-carnitine, creatine, and creatinine in real tear samples of healthy individuals (n = 7) were within the range of 1.1-8.2, 4.8-46.3, and 8-44.7 µmol/L, respectively. Obtained results were compared with published results in tears if available (L-carnitine and creatinine) or with other biological matrices. As our knowledge, this publication is the first that reports the content of creatine in tears.