Brief Communication Rapid detection of DEHP in packed red blood cells stored under European and US standard conditions Angelo D'Alessandro, Travis Nemkov, Kirk C. Hansen Department of Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, United States of America
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LC-mass spectrometry (MS) based methods have been proposed to detect phthalate esters and, in particular, DEHP in RBC stored in SAGM and AS-15,9,10 or in urine for autologous blood doping detection11,12. Here we propose an evolution of those methods using ultra-HPLC (UHPLC) and high resolution MS for the ultra-rapid detection (4 min vs 20 min and 10 min with the stateof-the-art methods5,13) to quantify DEHP in RBC stored in SAGM and AS-3. We also explore the potential of this method to detect additional compounds of interest, such as MEHP and phthalate catabolites.
Materials and methods
DEHP standard: linearity, sensitivity, reproducibility, recovery and matrix effects DEHP was quantified using a commercially available standard (#101225813 - Sigma, St Louis, MO, USA). Standard (0.985 g/mL) was sequentially diluted in lysis/ extraction buffer (methanol:acetonitrile:water 5:3:2 v/v; 10 µM 5-fluorouracil [5-FU] as internal standard) until limits of detection (signal 3 times above noise) and limits of quantitation (last linear point of calibration curve at least 3×limit of detection) were achieved. Samples were run on an UHPLC system (Ultimate 3000RS, Thermo Scientific, San Jose, CA, USA) through a Kinetex C18 column (150×2.1 mm internal diameter, 1.7 µm particle size - Phenomenex, Torrance, CA, USA) using a 9 min gradient (5% B for the first 2 min; 5-95% B over 1 min; hold at 95% for 2 min; 95-5% B over 1 min; re-equilibrate for 3 min) or an isocratic 4 min run at 95% B (mobile phases: A=18 mΩ H2O, 0.1% formic acid; B=acetonitrile, 0.1% formic acid). The UHPLC system was coupled online with a QExactive tandem mass spectrometer (Thermo Scientific), scanning in Full MS mode at 140,000 resolution in the 60-900 m/z range, operated in positive ion mode. Calibration was performed before each analysis using positive ion mode calibration mixes (Piercenet - Thermo Fisher, Rockford, IL, USA) to ensure sub-ppm error of the intact mass. We tested linearity over seven orders of magnitude (concentrations from nmol to low fmol range) for the 9- and 4-min methods. Based on the positive results of the 4-min method, reproducibility was assessed across three non-consecutive days, using the spiked in internal
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Transfusion of packed red blood cells (RBC) is a lifesaving intervention for millions of people worldwide. Thorough statistical review of 31 randomised clinical trials concluded that, while "restrictive transfusion strategies are safe in most clinical settings, liberal transfusion strategies have not been shown to confer any benefit to patients but have the potential for harm"1. Though randomised clinical trials performed so far have not highlighted a major association between storage duration and mortality, the studies may have been underpowered to appreciate minor (
Sr l
LC-mass spectrometry (MS) based methods have been proposed to detect phthalate esters and, in particular, DEHP in RBC stored in SAGM and AS-15,9,10 or in urine for autologous blood doping detection11,12. Here we propose an evolution of those methods using ultra-HPLC (UHPLC) and high resolution MS for the ultra-rapid detection (4 min vs 20 min and 10 min with the stateof-the-art methods5,13) to quantify DEHP in RBC stored in SAGM and AS-3. We also explore the potential of this method to detect additional compounds of interest, such as MEHP and phthalate catabolites.
Materials and methods
DEHP standard: linearity, sensitivity, reproducibility, recovery and matrix effects DEHP was quantified using a commercially available standard (#101225813 - Sigma, St Louis, MO, USA). Standard (0.985 g/mL) was sequentially diluted in lysis/ extraction buffer (methanol:acetonitrile:water 5:3:2 v/v; 10 µM 5-fluorouracil [5-FU] as internal standard) until limits of detection (signal 3 times above noise) and limits of quantitation (last linear point of calibration curve at least 3×limit of detection) were achieved. Samples were run on an UHPLC system (Ultimate 3000RS, Thermo Scientific, San Jose, CA, USA) through a Kinetex C18 column (150×2.1 mm internal diameter, 1.7 µm particle size - Phenomenex, Torrance, CA, USA) using a 9 min gradient (5% B for the first 2 min; 5-95% B over 1 min; hold at 95% for 2 min; 95-5% B over 1 min; re-equilibrate for 3 min) or an isocratic 4 min run at 95% B (mobile phases: A=18 mΩ H2O, 0.1% formic acid; B=acetonitrile, 0.1% formic acid). The UHPLC system was coupled online with a QExactive tandem mass spectrometer (Thermo Scientific), scanning in Full MS mode at 140,000 resolution in the 60-900 m/z range, operated in positive ion mode. Calibration was performed before each analysis using positive ion mode calibration mixes (Piercenet - Thermo Fisher, Rockford, IL, USA) to ensure sub-ppm error of the intact mass. We tested linearity over seven orders of magnitude (concentrations from nmol to low fmol range) for the 9- and 4-min methods. Based on the positive results of the 4-min method, reproducibility was assessed across three non-consecutive days, using the spiked in internal
©
SI
M
TI
Se
rv
Transfusion of packed red blood cells (RBC) is a lifesaving intervention for millions of people worldwide. Thorough statistical review of 31 randomised clinical trials concluded that, while "restrictive transfusion strategies are safe in most clinical settings, liberal transfusion strategies have not been shown to confer any benefit to patients but have the potential for harm"1. Though randomised clinical trials performed so far have not highlighted a major association between storage duration and mortality, the studies may have been underpowered to appreciate minor (
