Development and validation of a robust UPLC-MS/MS method for the analysis of polyamines in cells, biofluids and...

Loss of function (LoF) of the ATP13A2 protein, a polyamines transporter, has been linked to lysosomal and mitochondrial dysfunctions that play an important role in the early onset of Parkinson's disease (PD) and related neurodegenerative disorders. To investigate the downstream consequences of these LoFs in relevant in-vitro and in-vivo models, we describe the development, optimization, validation and application of an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of a range of endogenous polyamines (putrescine, cadaverine, ornithine, spermidine, spermine, N1 acetylputrescine, N1 acetylcadaverine, N1 acetylspermidine, N1N8 diacetylspermidine and N1 acetylspermine) in cells, biofluids and brain tissues. Sample pre-treatment consists of protein precipitation, derivatization with benzoyl chloride (BZ) and liquid-liquid extraction clean-up with ethyl acetate. The chromatographic separation is achieved on an ACQUITY BEH C18 column within 5.5 min using an acetonitrile/water + 0.1 % formic acid gradient, while the mass spectrometric analysis is performed using a triple quadrupole operating in selected reaction monitoring (SRM) mode. The analytical method is validated in terms of linear dynamic range, lower and upper limit of quantification (LLOQ and ULOQ), accuracy, precision, matrix effect, carry-over and stability. LLOQ values are between 0.05 and 0.5 ng/mL while the ULOQ values are between 1*10³ and 5*10⁴ ng/mL, depending on the analyte and on the matrix. For certain compounds, the linear dynamic range extension, up to four orders of magnitude, is achieved using isotopologue transitions. Accuracy is between 97.5 % and 108.2 % while intra-day and inter-day precision, expressed as cv% of the control samples at three different concentration levels, are below 15 %. No carry-over and matrix effect are observed. The analysis of cell lysates, different biofluids (mouse plasma and mouse cerebrospinal fluid) and mouse brain tissue homogenates confirm the applicability of the developed method for the quantification of the polyamines included in the method.