Introduction
Sample Preparation
Data Acquisition
DSS Concentration Measurement
Measuring T1 Relaxation Times

Accurately quantifying metabolites using targeted profiling in Chenomx NMR Suite requires using a chemical shape and shift indicator (CSI) of known concentration. As a result, the accuracy of the CSI concentration has a significant influence on quantification accuracy using this technique. Measuring ratios of the CSI peak area with that of a second, commercially available standard under optimal acquisition conditions offers a simple method of accurately measuring the concentration of the CSI.

Quantifying metabolites absolutely using targeted profiling in Chenomx NMR Suite requires using a chemical shape and shift indicator (CSI) of known concentration, usually 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) or 3-(trimethylsilyl)propionic acid (TSP).

This note describes a method for preparing and determining the concentration of a DSS solution for use as a CSI with Chenomx NMR Suite using 1D-1H NMR spectroscopy. The method requires the use of a second commercially available standard compound of certified concentration.

An appropriate commercially available compound should:

  • be soluble in the solvent of choice
  • be chemically stable

  • have at least one reference signal that:

    • does not overlap with signals from DSS or TSP
    • is not subject to proton exchange with the solvent
    • is not affected by solvent suppression techniques (where applicable)

In the following example we describe the experimental steps and NMR methods used by Chenomx to prepare and quantify a DSS solution using a commercially available sodium acetate NMR standard.

The chosen NMR reference standard (50 mM sodium acetate in 99% D2O, 500 μL) is combined with the DSS solution to be quantified (~5 mM DSS in D2O, 500 μL) and vortexed for 30 seconds. 600 µL of the resulting solution is transferred to an NMR tube for analysis. An NMR reference standard comprising a sodium acetate solution is available from ISOTEC, a member of the Sigma-Aldrich group, product #613053.

The concentration of DSS is determined using the 1D-1H NMR peak area ratio between the methyl signals of DSS and acetate. The NMR experiment must allow for complete longitudinal relaxation (T1) of both components. At 500 MHz, the T1 relaxation times of the methyl groups are 3.4 seconds for DSS and 6.1 seconds for acetate (see Measuring T1 Relaxation Times). Details of the pulse sequence used to acquire the quantitative experiment appear in Figure 1. Briefly, the pulse sequence consists of a 2 second recycle delay, then a single 1 µs hard pulse followed by an 8 second detection period.

Longer delays may be required at higher field strengths to ensure complete longitudinal relaxation. Using a shorter 1-µs pulse instead of a full 90° pulse reduces the signal-to-noise ratio in each scan, but allows a shorter recycle delay (more scans) over the duration of the experiment. The 90° pulse would require approximately 40 seconds (5 x T1) of recycle delay for accurate quantification and complete longitudinal relaxation.

500 MHz 1D-1H NMR pulse sequence used for DSS concentration determination in aqueous solution (D2O). Numbers in blue indicate timings in seconds (s); red indicates timings in microseconds (μs).

Figure 1. 500 MHz 1D-1H NMR pulse sequence used for DSS concentration determination in aqueous solution (D2O). Numbers in blue indicate timings in seconds (s); red indicates timings in microseconds (μs).


The acquired free induction decay (fid) is zero-filled and Fourier-transformed with no windowing function, and the resulting 1D-1H NMR spectrum is manually phased and baseline corrected. Baseline correction is performed using VNMRj (Varian) and an in-house modified version of the hregions macro that excludes the application of autophasing. Sodium acetate produces a simple 1H-NMR spectrum consisting of a single peak near 1.91 ppm, locating it away from the water resonance, and not overlapped with DSS (see Figure 2).

500 MHz 1D-1H NMR spectrum of sodium acetate (1.91 ppm, 25 mM) and DSS (0 ppm, 2.5 mM).

Figure 2. 500 MHz 1D-1H NMR spectrum of sodium acetate (1.91 ppm, 25 mM) and DSS (0 ppm, 2.5 mM).


Peak areas are integrated over a total width of 0.1 ppm centered at 0 ppm (ADSS) and at 1.91 ppm (Aacetate), and the concentration of DSS in the sample is calculated as:

Peak Area Ratio Equation: [DSS] = [acetate][A(DSS)/k*A(acetate)]

where [acetate] is the known concentration of acetate in the sample, and k is the ratio of the number of protons of the DSS peak (9) with the number of protons of the acetate peak (3). For the current application, k=3.

The T1 relaxation times at 500 MHz for the methyl groups of both acetate and DSS are measured using the T1meas pulse sequence (Varian) at 25°C in D2O. A total of ten "d2" time delays are arrayed on an exponential curve from 0.5 to 10 seconds (see Figure 3).

Intensity Decay Equation: I(t) = [I(t) - I(0)]exp(-t/Tsub1) + I(0)

Figure 3. T1 relaxation decay curves for the methyl groups of acetate (red) and DSS (blue) at 500 MHz and 25°C in D2O.


The intensity of the acetate and DSS methyl peaks were measured automatically with the dfp and t1 built-in macros (Varian), and the intensity decays were fit using Mathematica to the following equation:

Intensity Decay Equation: I(t) = [I(t) - I(0)]exp(-t/Tsub1) + I(0)
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