Absolute Quantification: Why qNMR is Important?
All biomarkers and internal standards from GelbChem are quantified by qNMR rather than gravimetrically.
As described below and recently published (1), quantitative nuclear magnetic resonance (qNMR) is critical to ensure that the absolute moles of reagent in each vial is known as precisely as possible. Typically, one wants to measure the absolute abundance of a metabolite in a biological sample. If this is done by MS/MS, one needs to use an internal standard that is chemically identical to the metabolite of interest but differentiated by substitution with a heavy isotope. At a minimum, it is required that the true concentration of the analyte in a stock solution be known as accurately as possible. Then one can inject an absolute number of moles of analyte into the instrument (the true moles) together with an aliquot of a solution of internal standard and determine the ratio of analyte to internal standard MS/MS response (response ratio). It is not required that the true moles of internal standard in the stock solution be accurately known. Even if a truly equi-mole mixture of analyte and internal standard are analyzed, the response ratio may not be 1 because of biases in the tuning of the quadrupole mass filters and/or an isotope effect on the fragmentation of the parent ion to give the product ions (no isotope effect is expected if a bond to the heavy isotope does not break in the fragmentation reaction). Thus, the measured response ratio will reflect the true difference in absolute moles of the analyte and internal standard analyzed, tuning bias, and any isotope effects on fragmentation. For example, if one injected truly 1 nmole of an analyte and nominally 1 nmole of internal standard in the MS/MS instrument and observed a response ratio of 1.2, one can obtain the true moles of analyte in a new sample via the following equation:
moles of analyte in sample = [(measured analyte-to-internal standard MS/MS response ratio) x (moles of internal standard in the sample)] divided by 1.2
The above equation is valid even if the absolute moles of internal standard in the stock solution (and thus added to the sample) is not the true value as long as the same internal standard stock solution is consistently used for all samples, and the absolute moles of analyte in the stock solution used to determine the response ratio is the true value. If the true moles of internal standard in the stock solution is also known, one can measure the effect of tuning bias and fragmentation isotope effect on the response ratio, but this is not required. The challenge comes in knowing the true moles of analyte in the stock solution as discussed next.
Gravimetric analysis. Many reference laboratories rely on gravimetric methods in which a microbalance (accurate to say 0.1-1 mg) is used to weight 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. For example, 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). High pressure liquid chromatography may be used in cases of molecules that absorb UV light. However, these methods cannot establish that the standard compound is pure by weight. For example, 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. Without knowing the weight purity of the standard compound, one cannot know the true moles of standard compound in the stock solution that was made gravimetrically.
Purity by weight and qNMR. Purity by weight may be assessed by combustion analysis in which the amounts of CO2, H2O and NO2 are measured and compared to the amounts of carbon, oxygen, and nitrogen calculated based on the molecular formula of the standard. This method is rarely carried out on heavy isotope-substituted internal standards because of the relatively large amounts of material needed. The residue after burning is also used, but again requires large amounts, and has some pitfalls. Arguably, the most appropriate method to determine the true moles of standard in a container is quantitative proton nuclear magnetic resonance (qNMR). This technique is well known to most analytical chemists, and it relies on the ability of qNMR to provide a count of the number of hydrogen atoms in the sample tube. As an example, consider the sphingolipid psychosine (Figure 1), which is a useful biomarker for the diagnosis of Krabbe disease. The qNMR is shown in Figure 1. A series of peaks are seen in this spectrum that reflect the different kinds of hydrogen atoms, and assignment of each peak to the various hydrogens in psychosine is easily done using reference tables of NMR data from countless molecules over the past half-century. For example, psychosine has one double bond, and each of the hydrogens attached to the doubly-bonded carbons can be found in the NMR at a specific chemical shift position along the X-axis (Figure 1). Also seen are peaks due to the hydrogens of the internal standard N,N-dimethylformamide (DMF). Internal standards used for qNMR are simple organic molecules that are reliably obtained from commercial sources in near 100% purity by weight. They are usually purified by distillation and thus free of non-volatile impurities such as salts and metals. The possible presence of impurities that could co-distill with DMF can be ruled out by the absence of spurious NMR signals (signals other than those from the hydrogens of DMF). Since DMF is taken as 100% pure by weight with high confidence, the moles of this standard in the NMR sample tube along with psychosine are known by adding an accurate weight or volume of DMF to the tube. The nice feature of qNMR is that the area under each signal peak is proportional to the number of hydrogen atoms in the molecule. For example, if the number of molecules of psychosine in the NMR tube is equal to the number of DMF molecules in the same tube, the area under the NMR peak due to the methyl group of psychosine (3 hydrogens) will be 3-times that under the NMR peak of DMF due to its single formyl proton (Figure 1). One may worry that there are impurities (non psychosine molecules) in the sample that contribute to peak area for say the psychosine methyl group (buried in the methyl peak), but the ratio of peak areas for the various NMR peaks of the psychosine hydrogens can be compared to each other and with those predicted from the chemical structure of this molecule (Figure 1) (the same is true for the internal standard).
A good description of the qNMR experiment is available at: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Brochure/1/qnmr-brochure-rjo.pdf. 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.
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.
1. M H Gelb, (2018) "Absolute Amounts of Analytes: When Gravimetric Methods Are Insufficient." Clin. Chem. 64: 1430-32.