| IntroductionPolyamines are found widely in living organisms,which are long-chain aliphatic amines,mainly including putrescine,spermidine and spermine.They have long been closely associated with cell growth,tissue growth and cancer.It has been reported in a large number of literature that polyamine levels were remarkably increased in the body fluids,tissue and hair of patients with cancer.It is well-established that polyamine may serve as a diagnostic marker for patients with cancer.In our study,solid-phase extraction(SPE)using diatomaceous earth columns followed by derivatization with HFB-C1 and elution with volatile n-hexane simultaneously proved to be available and rapid by simplifying the whole procedure of samples preparation.The method was successfully applied to the analysis of 36 hair samples from 14 healthy men and 22 healthy women by gas chromatography-mass spectrometry(GC-MS).MethodsEach of hair sample(50 mg)was extracted with 1 mol/L NaOH solution together with I.S.,at 95℃for 1.5 h in a water bath.After cooling,hair samples were centrifuged at 3000 rpm for 10 min after adjusting to pH＜1 with concentrated hydrochloric acid. The aqueous layer was poured onto the solid-phase extraction(SPE)column filled with 1.7 g of desiccated diatomaceous earth after adjusting to pH 12-13 with 5 mol/L NaOH solution for the reaction of the basic amino functions.After 10 min for equilibration, the analytes were eluted with a mixture of n-hexane,butyl acetate and HFB-C1.The eluent was collected in a glass tube and evaporated down to ca 30μl under a stream of nitrogen followed by GC-MS analysis.ResultsThis method was linear,reproducible,accurate and sensitive.The sharp and symmetrical peaks of four polyamines were obtained without disturbance of endogenous interferences.The method was successfully applied to the analysis of 36 hair samples from 14 healthy men and 22 healthy women.The three polyamines were positively detected in aliquots of 50 mg of all hair samples.Either in the male or female group,the most abundant hair polyamine was spermidine,followed by putrescine and spermine,while the mean levels of the three polyamines in hair samples were all found to be higher in men than in women.DiscussionThere are some advantages of hair as biosample for determination polyamines, such as non-invasive,stability of endogenous components,reflecting long-term chronic biochemical information and convenient collection and storage.Recently,the value of hair as the non-invasive biosample for determination endogenous compounds has been recognized in the biomedical field.In our preliminary experiment,acid hydrolysis and alkaline digestion were adopted to the hair pretreatmant,respectively.Results showed significantly lower polyamine concentrations in the same hair sample after acid hydrolysis compared to alkaline digestion.Diatomaceous earth was hydrophilic filler and its mechanism of action was distribution principle.The dosage of diatomaceous earth must match the usage amount of supernatant.The eluent in this study was n-hexane.ConclusionThe GC-SIM-MS method developed in this study has been shown to be suitable with acceptable reproducibility and recoveries for simultaneous determination of putrescine,spermidine and spermine as N-HFB derivatives in hair samples.Advantages offered by the method were simple preparation of hair samples,effective separation, high sensitivity and short time analysis.The present validated method for measuring hair polyamines may be assistant in early diagnosis and prevention of cancer in patients. Moreover,this study applied for determination polyamines in other biological samples is underway. |
PDF Full Text Request