Dear Samyr,

You may use the following protocol:

Extraction and derivatization protocols

Polyamine extraction from biological sources is commonly performed by acid extraction with cold TCA or perchloric acid (PCA) with subsequent neutralization for analysis of cultured cells [33] and animal and plant tissue [34]. In general, homogenization is required for tissue, and a variety of standard techniques can be used (for a set of references see [34]). For tissues that are difficult to homogenize (e.g. plant tissues or chitin-rich insects), a protocol involving repeated freeze–thaw cycles may be beneficial, especially when larger sample numbers need to be processed [34]. Based on these methods [33,34], the following protocols were developed for the polyamine analyses described in this paper. Extraction protocols for serum, tissue, cell and whole-blood samples are summarized in Fig. 1. For tissue, cell or whole-blood samples, an individual dilution step was required before sample preparation, which enabled polyamine concentrations to fall within the linear calibration range. Table 1 shows the expected concentrations of polyamines in various tissues, organism or cells. Based on this concentration range, samples were individually diluted by using the dilution factor Dspecific for tissue, cell or whole blood. This dilution factor D was calculated for the analytes put, spd and sp. Sample dilution was always performed by increasing the volume of TCA and IS. D is calculated for 30 mg tissue or 107 cells or 20 μl whole-blood samples. For orn, no dilution was required, since the concentration of orn in different samples was always within the calibration range of orn. The derivatization of polyamines was carried out according to the method of Byun et al. [21]. Amine carbamoylation derivatization of polyamines was performed by using isobutyl chloroformate. The derivatization protocol is presented in Fig. 1.

Chromatographic methods

All experiments were carried out on an Ultimate 3000 HPLC system comprising an autosampler and a 10-way switching valve unit coupled to a triple-quadrupole mass spectrometer, a Quantum TSQ Ultra AM, both from Thermo Fisher Scientific (San Jose, CA). The system was controlled by Xcalibur Software 2.1. The oven of the autosampler was maintained at room temperature, and the tray at 5 °C. For online SPE–LC/MS/MS, two Strata-X SPE cartridges (2 × 20 mm, 25 μm particle size) from Phenomenex (California, USA) were installed. Additionally, an in-line filter (KrudKatcher Classic HPLC In-Line Filter, 0.5 μm Depth Filter, Phenomenex (California, USA)) was placed in front of the 10-way switching valve unit to protect both SPE columns.

For more information, please use the following link:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991419/

Another publication which describes the required protocol can be found in the following link:

http://www.ncbi.nlm.nih.gov/pubmed/24633404

Hoping this will be helpful,

Rafik

Dear Samyr,

This is a site on an article on: Determination of total polyamines in tumor cells by high-performance capillary zone electrophoresis (HPCZE) with indirect photometric detection

Rulin Zhang, Cynthia L. Cooper, Yinfa Ma

Anal. Chem., 1993, 65 (6), pp 704–706

DOI: 10.1021/ac00054a009

Publication Date: March 1993

https://www.google.com.eg/url?sa=t&rct=j&q=&esrc=s&source=web&cd=4&cad=rja&uact=8&ved=0ahUKEwiXsZjxicnOAhVHiRoKHSqECh8QFggyMAM&url=http%3A%2F%2Fpubs.acs.org%2Fdoi%2Fpdf%2F10.1021%2Fac00054a009&usg=AFQjCNGC7vB9yU3-gcm_MISeEMegd3lmjA

With my best wishs

Dear Samyr,

Please, find in attach a paper on: Optimization of dansyl derivatization and chromatographic conditions in the determination of neuroactive amino acids of biological samples

Xuejun Kang a,c,*, Jing Xiao c, Xiao Huang c, Zhongze Gu

It might be useful

Good luck

Check this article https://link.springer.com/content/pdf/10.1007%2Fs12257-011-0346-6.pdf