Tests D-I robustness

Test D Variation of pH value. Seven samples were prepared according to the standard operational procedure, but the pH value was varied by addition ofNa2C03 or HCl. 

Within the given limits, variation of the pH value had no influence on the results. The recommended pH value is 8.5, values above 9.5 and below 6.5 lead to hydrolysis of the phospholipids. Additionally, the line shape of the NMR signals broadened under the more acidic conditions and the chemical shift showed large variations. 

Test E Variation of the ternary solvent mixture. According to the standard operation procedure for sample preparation equal amounts (by volume) of CDCI3, methanol and Cs-EDTA (caesium ethylenediaminetetraacetic acid) solution were used. Cs-EDTA was held constant at 1 ml, CDCI3 was changed from 1 ml to 0.6 ml and MeOH from 1 ml to 1.4 ml. 

There was no significant influence on the results of changing the composition of the solvent mixture within certain limits. Further changes can lead to line broadening and/or to reduced extraction yield. 

Test F Variation of the amount of sample. The amount of sample was varied from 60 mg to 140 mg, and the amount of internal standard between 20 mg and 40 mg, taking care that even extreme ratios were covered, from 6 4 to 7 2. 

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