

Absolute Quantification
by
qNMR
THE GOAL:
Determine the exact number of molecules (moles) in a sample
Why it Matters
Often, scientists need to know the exact amount of a specific molecule (called a metabolite) in a biological sample. This helps understand its role in biological processes.
The Challenge
Measuring absolute moles can be tricky. Techniques like Mass Spectrometry (MS/MS) rely on standards and internal standards, but the response (peak area per mole) for these standards can be affected by instrument settings.
USING INTERNAL STANDARDS
IN MASS SPECTROMETRY
An internal standard is a molecule structurally similar to the one you're measuring (the metabolite) but modified in some way to differentiate it (often by substituting atoms with heavy isotopes).
What it is
The ratio of the response (peak area) from the metabolite over the response of the internal standard is used to determine the actual amount of the metabolite present.
Why it's used

The response factor is the signal from the metabolite compared to the internal standard in the MS/MS analysis. Even with equal amounts of metabolite and internal standard, the response ratio might not be 1 due to instrument tuning or how the molecules break down in the analysis (isotope effect).
The Response Factor may not be 1
CALCULATING TRUE MOLES OF A METABOLITE/BIOMARKER
With the measured response ratio and known moles of internal standard, you can calculate the true number of moles of the metabolite in a new sample using the following equation:

You need to know the exact concentration of the metabolite in its stock solution. This ensures accurate injection into the instrument for comparison with the internal standard.

THE TROUBLE WITH GRAVIMETRIC ANALYSIS
Many reference laboratories rely on gravimetric methods in which a microbalance (accurate to say 0.1-1 mg) is used to weigh out a few milligrams of a commercial standard compound that is reported to have a high purity (say > 99%).. However, it is critically important to investigate how purity is measured.
It is critically important to investigate how purity is measured.
EXAMPLE 1:
TLC
Purity of lipid standards is often based on thin layer chromatography in which lipids are visualized with a stain that binds to hydrophobic molecules (i.e., iodine staining). This type of purity analysis is limited by hydrophilic impurities it cannot detect.
EXAMPLE 2:
UV ABSORPTION
High performance liquid chromatography (HPLC) may be used in cases of molecules that absorb UV light. However, these methods cannot establish that the standard compound is pure by weight.
EXAMPLE 3:
TYPES OF IMPURITIES
Most chromatographic methods will not detect impurities such as water, silica gel (often left over from chromatographic purifications), salts, and metals, just to name a few.
qNMR: A RELIABLE METHOD
Quantitative Nuclear Magnetic Resonance (qNMR) is a technique that counts the number of hydrogen atoms in a sample.
qNMR uses internal standards (like DMF) known to be nearly 100% pure by weight to measure the number of molecules per gram of sample.
What it is
Each hydrogen atom in the molecule contributes to a specific signal in the qNMR spectrum. By comparing the signal areas of the metabolite and the internal standard, one can calculate the relative number of molecules present.
How it Works

Figure 1. qNMR spectrum of psychosine in deuterated methanol (CD3OD) containing DMF as an internal standard. The Y-axis is NMR signal intensity in arbitrary units, and the X-axis is the chemical shift (f1) in units of parts per million (ppm). The peak at 8.0 ppm is from the formyl hydrogen of DMF (H-CON(Me)2), and the peaks at ~2.85 and ~3.00 ppm are due to the methyl groups of DMF. The number below each peak is the peak area; note that the methyl peaks of DMF are ~3-fold the area of the formyl peak (which was assigned to 1.00 for convenience). The peaks at ~5.55 and ~5.90 ppm are due to the hydrogens attached to the double bond of psychosine, the peak at ~0.90 ppm is due to the methyl group, and the peak at ~2.20 ppm is due to the CH2 next to the double bond. Other peak assignments are known but not indicated in the Figure. Note that the area ratios are very close to those expected based on the structure of psychosine except that the peak at ~5.55 is of a slightly higher area than the peak at ~5.90 ppm suggesting a small amount of impurity that contributes proton area to the ~5.55 ppm peak. By using these areas and those of the DMF internal standard and knowing the absolute moles of DMF in the tube (based on gravimetric analysis of DMF that is essentially pure by weight), one obtains the absolute moles of psychosine in the sample. This is valid even if the psychosine contains impurities that lower its purity by weight. If the moles of psychosine determined by qNMR agrees with the moles measured gravimetrically, only then can it be said that psychosine is pure by weight.

BENEFITS OF qNMR
qNMR minimizes errors from impurities in the standard compound.
Accuracy
qNMR assigns signals to specific hydrogen atoms in the molecule for further analysis.
Specificity
A good description of the qNMR experiment is available by clicking the button below. This document describes the instrument settings for a reliable qNMR experiment including the important need for a longer than normal recycle delay between radio-frequency pulses. The latter is sometimes overlooked by NMR operators.
More Information
1. M H Gelb, (2018) "Absolute Amounts of Analytes: When Gravimetric Methods Are Insufficient." Clin. Chem. 64: 1430-32.
Reference