Development of exposure assessment method based on the analysis of urinary heterocyclic amines as biomarkers by on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry

Abstract

Heterocyclic amines (HCAs) formed in cooked meats and fish are mutagens and carcinogens in rodents and nonprimates. Exposure to HCAs may also be a risk factor for human tumors, but the association between dietary intake and human cancer risk has not been determined. To assess recent exposure to HCAs, we developed a simple and sensitive method for measuring HCAs in urine by automated on-line in-tube solid-phase microextraction (SPME) using a Supel-Q PLOT capillary column as an extraction device, in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Thirteen HCAs were separated within 15 min using a ZORBAX Eclipse XDB-C8 column and detected selectively by multiple reaction monitoring using MS/MS. This method can be applied easily to the analysis of small amounts of urine samples without any other pretreatment except for alkaline hydrolysis of bound forms of HCAs. The quantification limits of HCAs in 0.2 mL of urine samples were about 1.7-4.1 pg/mL (S/N = 10). Using this method, we evaluated the exposure to HCAs in persons who consumed well-done pan-fried beef and the suitability of using urinary HCAs as exposure biomarkers. We also analyzed the ability of vegetable consumption to prevent carcinogenic risks from exposure to HCAs by measuring free and bound forms of HCAs in urine.

Biomarkers of meat and seafood intake: an extensive literature review

“…Other compounds besides PhIP and MeIQx have been identified in urine after the intake of roasted meat (Additional file 2: Table S3). These include 9 H -pyrido[3,4- b ]indole (norharman) [132, 138], IQ (2-Amino-3-methylimidazo[4,5-f]quinoline), Trp-P-2 (3-amino-1-methyl-5 H -pyrido[3,4- b ]indole), Trp-P-1 (3-amino-1,4-dimethyl-5 H -pyrido[3,4- b ]indole), and AαC (2-amino-9 H -pyrido[2,3- b ]indole) and harman [138]. However, these molecules were found to be less reliable as markers of exposure to heated meat, when compared to PhIP and MeIQx.…”

High-throughput and sensitive analysis of urinary heterocyclic aromatic amines using isotope-dilution liquid chromatography–tandem mass spectrometry and robotic sample preparation system

“…Recently, automatic, on-line, in-tube, solid-phase microextraction (SPME) was used to increase the throughput of urinary HCAA analysis. [26] Compared with previously reported manual methods, SPME significantly improved analytical throughput, but at the cost of higher background noise in the chromatogram. [26] In the present study, a two-step SPE with multiple intensive washing procedures was used to minimize chromatographic background noise from matrix.…”

“…[26] Compared with previously reported manual methods, SPME significantly improved analytical throughput, but at the cost of higher background noise in the chromatogram. [26] In the present study, a two-step SPE with multiple intensive washing procedures was used to minimize chromatographic background noise from matrix. At the same time, we enhanced throughput via use of a robotic automation system (Perkin Elmer Staccato system) for sample preparation and increased the sensitivity by using API 5500 QTRAP LC-MS/MS for sample analysis.…”

Bioanalytical HPLC Applications of In-Tube Solid Phase Microextraction: A Two-Decade Overview

“…This problem was overcome with the use of Carboxen 1006 PLOT column, which also showed great extraction efficiency [31]. Supel Q PLOT columns have been successfully employed for the in-tube SPME of various organic compounds, including heterocyclic amines from hair [36], endocrine disruptors from liquid medicines and intravenous injection solutions [51], melatonin from saliva [52], cortisol from human saliva [53], urinary heterocyclic amines [54], testosterone, cortisol, and dehydroepiandrosterone from saliva [55], and anabolic steroids from urine [56].…”

“…Compared to the conventional WCOT liquid-phase GC columns, PLOT columns exhibit a larger adsorption surface and thicker film layer, resulting in higher extraction efficiency of the target analyte. Various PLOT columns have been evaluated, including Carboxen 1006 PLOT capillary column (carbon molecular sieves) (Supelco) [31,32,[48][49][50], Rt-U PLOT (divinylbenzene ethylene glycol/dimethylacrylate) (Restek (Bellefonte, PA, USA) [38], CP-Pora PLOT amine (basic modified stylene divinylbenzene polymer) (Varian Inc., Lake Forest, CA, USA) [48], and Supel Q PLOT (Divinylbenzene polymer) (Supelco) [32,36,48,[51][52][53][54][55][56].…”