Chapter 3 52 Sample preparation EDTA whole blood samples were collected between August 2017 until December 2017. One aliquot sample was prepared for iIEM and one for dIEM. Erythrocytes were isolated from plasma according to a strict protocol to obtain washed erythrocytes for dIEM. First, plasma and buffy coat layer were removed after centrifugation of EDTA whole blood samples at 428 x g for 10 minutes at 4℃. The erythrocytes were washed using phosphate buffered saline (PBS). Samples were centrifuged (428 x g for 10 minutes at 4℃) to separate the PBS from the erythrocytes. Finally, the supernatant PBS was removed after centrifugation. These steps were then performed again, so that each sample was washed twice with PBS. After washing, a part of the packed red blood cells was used for an erythrocyte count. The rest of the packed red blood cells were stored at -80℃ until thawed for testing. IEM assay Magnesium can be accurately quantified using a variety of analytical techniques, including, but not limited to, atomic absorption spectrophotometry, particle-induced x-ray emission and inductively coupled plasma mass spectrometry (ICP-MS) 21. An advantage of ICP-MS over other analytical techniques, such as atomic absorption spectrophotometry, is the lower limit of the detection 22. In the present study, a validated ICP-MS method was used to determine magnesium in whole blood (for iIEM) and washed erythrocytes (for dIEM). The measurement of magnesium in serum and whole blood by an ICP-MS method has been previously validated 23. However, the measurement of IEM on an ICP-MS has not been validated before. Here, we describe the validation of magnesium in whole blood and washed erythrocytes on an ICP-MS in short that was performed at the University Medical Center Groningen. After mixing, 50µl sample was diluted to 5ml using a dilution reagent consisting of 0.1 mg/L Yttrium and 0.05% Triton-X100 and 0.05% EDTA in water. The diluted sample was measured with a Varian 820-ms ICP mass spectrometer. Six calibration standards, ranging from 5-50 mg/L were used and 3 quality control samples were measured with each run. The lower limit of quantitation was set to 5 mg/L. All measured samples were above the lower limit of quantitation. There were no matrix effects for whole blood or washed erythrocytes (equal slopes in different matrices) and calibration curve correlations were >0.999. There were no significant interferences of other elements during the analyses. Accuracy was assessed at 4 concentration levels using Magnesium ICP-MS standard (VWR Chemicals, Radnor, Pennsylvania, US). For each concentration level, standards were measured 18 times over the course of 3 days. Bias was -3,2% at 5 mg/L, 2,4% at
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