Rapid LC-MS/MS quantification of cancer related acetylated polyamines in human biofluids

Abstract

Increased urinary acetylated polyamines (APs) are reported as cancer biomarkers in many studies. N 1 ,N 12-diacetylspermine has been proposed as a biomarker indicative of different cancers in urine and plasma. N 1-Acetylspermine has previously been found to be increased in the saliva of patients with breast cancer; however, in plasma this metabolite was too low abundant to be detected by previous analytical methods. In addition, no method has been reported to perform AP analysis on the level of speed, robustness and sensitivity required for daily clinical routines. Here we describe a high-throughput sample preparation and LC-MS/MS method for the fast, accurate and precise quantification of three APs: N 8-acetylspermidine, N 1-acetylspermine, and N 1 ,N 12-diacetylspermine in plasma, urine and saliva. Stable isotope labeled N 1 ,N 12diacetylspermine was used as internal standard. Robustness was validated by intra-and inter-day reproducibility. Precision and accuracy of the method were tested at six concentration levels from 0.0375 to 750 ng/mL resulting in less than 15% relative standard deviation and less than 15% percent error in quantification. Using 96-well plates, the assay described herein allows for preparing, analyzing, and quantifying 240 samples per day for a single researcher to quantify three APs commonly related to cancer status.

Analytical Methods for Quantification and Identification of Intact Glucosinolates in Arabidopsis Roots Using LC-QqQ(LIT)-MS/MS

“…This process was repeated at three concentrations levels over three consecutive days to test inter-day and on the same day for intra-day precision and accuracy. The accuracy was expressed as the mean relative error percentage (%RE) and was calculated with the following formula: (mean of concentration found − added concentration)/added concentration × 100 [ 42 , 43 ]. Precision was expressed as the relative standard deviation percentage (%RSD) and was calculated by dividing the standard deviation with the mean of detected concentration and multiplying by 100 [ 42 ].…”

“…The accuracy was expressed as the mean relative error percentage (%RE) and was calculated with the following formula: (mean of concentration found − added concentration)/added concentration × 100 [ 42 , 43 ]. Precision was expressed as the relative standard deviation percentage (%RSD) and was calculated by dividing the standard deviation with the mean of detected concentration and multiplying by 100 [ 42 ]. Extraction efficiency (recovery) was calculated by dividing the mean peak area of glucosinolate standards spiked before extraction by the respective mean peak area of glucosinolate standards spiked to the blank matrix post-extraction and multiplying by 100 [ 42 , 44 ].…”

“…Precision was expressed as the relative standard deviation percentage (%RSD) and was calculated by dividing the standard deviation with the mean of detected concentration and multiplying by 100 [ 42 ]. Extraction efficiency (recovery) was calculated by dividing the mean peak area of glucosinolate standards spiked before extraction by the respective mean peak area of glucosinolate standards spiked to the blank matrix post-extraction and multiplying by 100 [ 42 , 44 ]. The matrix effect was calculated by comparing the mean peak area of the glucosinolates spiked into blank sample extract with peak areas of glucosinolates, which were prepared in the standard solvent.…”

Polyamines in Food

“…In fact, increased levels of acetylated polyamines in urine or blood, particularly, N1,N12-diacetylspermine, N1,N8-acetylspermidine, N1-acetylspermine, and N8-acetylspermidine have been found in patients with ovarian, prostate, colorectal, pancreatic, breast and lung cancers. Among them, N1,N12-diacetylspermine has been extensively described as the most effective urinary biomarker for several types of cancer and to monitor tumor's progression (48, 51, 52).…”

“…Post-column derivatization has the advantage that it is automatically performed online, thereby avoiding sample manipulation and shortening the time required for the analysis (89). In recent years, the determination of polyamines through liquid chromatography coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) has emerged as an alternative analytical technique, very specific and sensitive and without the need of derivatization (52, 87, 88, 90).…”