In order to perform optimally, it is best if the sample preparation and the NMR system for EDU data acquisition are performed in a manner similar to that used to prepare and collect the reference NMR system for research spectra used in Bayesil’s spectral library. Using different spectral preparation and collection conditions will compromise the performance, particularly the quantification accuracy. Here we describe the recommended methods that Bayesil users should follow when preparing biological samples and collecting NMR spectra of those samples.
All samples should have been either stored frozen or stored at 4 °C prior to use. Cerebrospinal fluid should be spun down to remove particulate matter and its pH checked (or adjusted) to ensure that it is between pH 6.8-7.4. More attention must be paid for the sample preparation of serum or plasma because of the high concentration of proteins in these samples. Proteins in the biofluid sample of interest must be removed by passing it through a pre-rinsed (washed 7X) Amicon Ultra-0.5 3000 MWCO filter at 4 °C using a centrifuge (12,000 g force). Unwanted protein signals are particularly problematic with serum and plasma and ultrafiltration is currently the best method of removing these signals without altering the chemical composition of the biofluid. After the protein filtration step has been completed, the sample may be placed into a NMR tube. If Shegemi tubes are used, 285 μL of sample will be needed. For regular thin walled 5 mm NMR tubes, 570 μL of sample will be needed. With respect to pre-rinsing the Amicon filters, sterile double-distilled water or its equivalent should be used. Rinsing should be done at least seven times to ensure any residual glycerol is removed. Otherwise glycerol concentrations will be erroneously elevated in the biofluid sample.
It is recommended that users check the pH of each sample prior to spectrum collection to ensure the pH is near 7.0. Otherwise the global fitting routine will produce larger errors than reported. The physiologic pH of blood and CSF is between 7.3 and 7.4. Plasma and serum generally have large buffering capacities, thus pH adjustment is rarely problematic. Bayesil is designed to work well with samples having a pH range between 6.8 and 7.5. However, the pH may vary due to sample handling or disease state and adequate optimization of the pH prior to data collection cannot be stressed enough. Larger concentrations of sodium phosphate than 50 mM may be necessary if your sample’s pH is not adequately controlled. Thus, checking your sample’s pH beforehand can alleviate many potential problems.
The necessary volumes and buffer recipes required for sample preparation are shown below in Table 1 and Table 2 respectively.