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According to the instruction from the kit manufacturer, 10 L of phosphate-buffered saline (PBS), calibrators, quality controls (QCs), and 10 L of tear samples were added to a 96-well plate which contains isotopic-labelled internal standards, dried under nitrogen at room temperature for 30 mins, followed by adding 50 L of phenylisothiocyanate (PITC) to derivatise amino acids and biogenic amines