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Calibration by NMR for quantitative analysis: p-Toluenesulfonic acid as a reference substance

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Abstract

We have demonstrated the usefulness of p-toluenesulfonic acid (TsOH) as a reference substance to calibrate a water-soluble standard by NMR for quantitative analysis. In order to make TsOH convenient for this purpose, we first of all established its molar extinction coefficient under these conditions (ɛ = 351 at 262 nm). This was done by comparing it by quantitative NMR (qNMR) with a compound of known molar extinction coefficient, guanosine monophosphate. TsOH was then used as the standard in a second qNMR experiment to measure an aqueous solution of phosphoglycolate, a metabolite of interest in our laboratory. Perhaps due to its content of water, the purity of this compound was found to be 82%, lower than the value of 94% from the manufacturer.

Introduction

As has been reviewed [1], [2], [3], [4], [5], quantitative nuclear magnetic resonance (qNMR) is a well established technique. Applications take place in many areas such as natural products [3], foods and beverages [6], metabolites [7], pharmaceutics [4], synthetic and combinatorial chemistry [2], commercial products such as gasoline [8], and agriculture [9].

qNMR experiments are increasingly needed in chemical analysis of biological samples, especially now that LC–MS has made it easier to measure complex analytes at trace levels. For calibration purposes, authentic samples of complex analytes (and corresponding internal standards) can be difficult to obtain, especially in higher amounts where absolute purity can be established more readily, and calibration then can be based on weighing. Even for higher amounts, the tendency of complex analytes to have an unknown content of water, solvent and salt complicates the calibration process. qNMR provides a way to help overcome these problems. This is because a small amount of a complex analyte can be measured independent of some contaminants in an absolute way by qNMR. From the calibrated sample, if devoid of UV-active contaminants, one can then immediately establish a molar extinction coefficient for more convenient calibration of future samples of a UV-active standard.

qNMR methods generally rely on comparing one or more NMR peak intensities of a compound of interest with a reference compound at a known concentration. The two compounds are usually co-dissolved for this comparison, but qNMR also has been done with separate solutions in a coaxial insert capillary tube [10], or via separate, sequential analyses on a given instrument [11]. qNMR can also be based on a reference electronic signal when appropriate instrumentation is available [12].

As has been reviewed [3], it is useful to have a diversity of reference compounds available for qNMR experiments in order to meet the needs of a given experiment. For a given analyte, the reference compound must be mutually soluble in an acceptable NMR solvent, provide a nonoverlapping signal relative to this analyte, and have contrasting physical properties when it is important to recover the analyte in a pure form afterwards. Ideally the reference compound can be calibrated by UV in order to avoid the errors associated with weighing.

Here we establish p-toluenesulfonic acid (TsOH) as a useful reference compound for some qNMR experiments. Its advantages (for some applications) are its high water solubility, commercial availability in a pure form, measurability in water by UV (based on this work), NMR signals in two resonance regions, and potential for facile removal from some compounds based on its physical properties. More specifically, we report the usefulness of TsOH as a NMR reference compound for calibration of phosphoglycolate, an analyte of interest in our laboratory as a product from oxidative damage to DNA.

Section snippets

Reagents

Phosphoglycolic acid tri(monocyclohexylammonium) salt (PGA, 99% aside from 4.9% H2O according to the supplier), p-toluenesulfonic acid monohydrate (>98.5%, A.C.S. reagent), and guanosine 5′-monophosphate disodium salt (GMP, 100% by HPLC, 19% loss on drying according to the supplier) were purchased from Sigma (St. Louis, MO, USA). Deuterium oxide (D2O, 99.9 atom% D) and organic solvents were from Fisher (Pittsburgh PA, USA).

Stock and assay solutions

Stock solutions (nominally 20 mM solute in each case) in D2O were

Results and discussion

We sought a convenient reference standard for quantitative NMR analysis of authentic DNA adducts in a nucleotide form which would have a strong resonance with a chemical shift of approximately 2 ppm (a region unoccupied in the NMR spectra of most nucleobases), along with a significant UV absorbance to facilitate accurate quantitation of a standard solution. TsOH was considered to meet these criteria, but we were unable to find a molar extinction coefficient for it in the literature. The single

Conclusions

TsOH is useful as a reference compound for quantitative NMR of aqueous solutions of compounds to be employed as authentic or internal standards in chemical analysis. Although this compound is not volatile, its subsequent removal from the compound of interest should be straight forward easy in most cases. TsOH should be similarly useful for calibration of standards that are soluble in organic-aqueous solutions.

Acknowledgement

This work was supported by NIH Grant CA 71993 received as a subcontract from Harvard Medical School. Contribution number 897 from the Barnett Institute.

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