Traceable Organic Certified Reference Materials for P-31 qNMR

Tools for the Quantification of Phosphorus

Phosphorus plays an important role in physiological processes since it is part of various significant molecules, such as nucleic acids (e.g. DNA), high-energy phosphates (e.g. ATP) and phospholipids. It is essential in the regulation of metabolism, since phosphorylation and dephosphorylation reactions are rapidly occurring processes at the protein level. Studies of physiological pathways, kinetics, metabolomics, and diseases, as well as biomarker discovery, are important fields of investigation, and there is a need for certified reference materials (CRMs) for the quantification of phosphorylated organic compounds and metabolites. The quantification by NMR offers several advantages as it is based on a signal comparison of the analyte with an internal or external reference standard. In contrast to other methods, such as chromatography, the reference standard is independent of the analyte’s chemical structure. Moreover, using very pure primary reference materials, qNMR is a highly accurate method with low measurement uncertainty, which also provides traceability to SI units (Système International d’Unités) and thus offers the possibility of certifying reference materials for ¹H or other nuclei, such as ³¹P.

This article presents the concept of value assignment by ³¹P qNMR measurements for the development of CRMs and describes different approaches to establish traceability to primary Standard Reference Material from the National Institute of Standards and Technology (NIST SRM) [1]. Water-soluble and phosphorus-containing CRMs are widely available, e.g. NIST SRM 194a (NH₄H₂PO₄), which can be used as a primary standard for establishing traceability in ³¹P qNMR measurements. Unfortunately, the situation is different for phosphorus-containing primary CRMs that are soluble in common organic solvents, such as dimethylsulfoxide or methanol. No internationally accepted reference standard, such as a primary CRM from a national metrological institute (NMI), is currently available, and therefore, direct traceability to a NIST SRM is not possible in the case of organic candidates. For that reason, the concept was to perform quantification via the protons using ¹H qNMR and use the assigned content to determine the purity of an analyte via the phosphorus using ³¹P qNMR. Phosphonoacetic acid is analyzed as a water-soluble CRM candidate, whereas triphenyl phosphate is a good candidate for use in organic solvents. The substances contain both nuclei, ¹H and ³¹P. For the validation of the concept, triphenyl phosphate and phosphonoacetic acid have been used as ³¹P qNMR standards to determine the purity of the analyte tris(2-chloroethyl) phosphate.

Preliminary tests have been performed to check the chemical compatibility between sample and internal standard by acquiring a proton spectrum and, where required, a ³¹P NMR spectrum of the mixture right after preparation and again after 24 h. It was necessary to exclude potential signal overlaps, as well as reactions between analyte and standard or reactions with the solvent. To ensure that no impurity was lying underneath the peaks of interest, 2D NMR experiments were applied where impurities of less than 0.05% signal intensity portion could be detected. Prior to the quantification measurements, the T1 relaxation times in different deuterated solvents were determined by inversion recovery experiments. Relaxation times should preferably be short, because the relaxation delay is adjusted to at least seven times T1. Since relaxation times may slightly vary in the mixture, the longest value in a mixture is used for calculating the overall relaxation time of the experiment. In a subsequent step, hygroscopy and volatility of the candidate substances were checked, since both attributes have a strong influence on the weighing results, and thus the outcome of the quantification measurement.

Quantification of the Candidate CRM Phosphonoacetic Acid

To prove the concept that no bias in a qNMR measurement is created when the purity is determined using either proton or phosphorus signals, the quantification of phosphonoacetic acid was performed by ³¹P and ¹H qNMR measurements . In the first experiment , the content of phosphonoacetic acid was determined by ³¹P qNMR using ammonium dihydrogen phosphate (NIST SRM 194a) as a reference, resulting in a purity value of 99.26% ± 0.75%. The second purity determination was carried out by ¹H qNMR measurement using potassium hydrogen phthalate (NIST SRM 84L) as a reference, resulting in a purity value of 99.32% ± 0.17%. These two results deriving from two independent traceability chains and via qNMR measurements of different nuclei are fully consistent and overlap within their expanded measurement uncertainties, as shown in Figure 4.

Quantification of the Candidate CRM Triphenyl Phosphate

After the ¹H and ³¹P qNMR experiments for the quantification of phosphonoacetic acid proved the independence of the purity value determined from the nucleus, triphenyl phosphate could be quantified by ¹H qNMR. Direct application of benzoic acid as standard reference material in proton NMR was not feasible, due to the overlapping of the signals in the aromatic region. Therefore, the traceability chain was achieved by the use of the CRM dimethyl terephthalate (07038) as an internal reference standard that is traceable to NIST SRM 350b, benzoic acid. The content of triphenyl phosphate was determined to be 99.95% ± 0.22%. Although this value was achieved by ¹H qNMR measurements, it can be employed in subsequent ¹H and ³¹P qNMR measurements as proven above. To remove any doubt regarding this conclusion, the concept was proven in a further step, in which a sample containing protons and phosphorus was chosen as the sample for the quantification by the two ³¹P qNMR CRM candidates.

Quantification of Tris(2-Chloroethyl) Phosphate by ³¹P qNMR

In the certification concept for phosphorus standards described, the chosen candidate compounds contain both ¹H and ³¹P nuclei. After triphenyl phosphate and phosphonoacetic acid have been quantified via their protons and/or phosphorus, the resulting purity values were used for the subsequent purity determination by ³¹P qNMR experiments. The quantification of tris(2-chloroethyl) phosphate on the basis of ³¹P qNMR yielded purities of 98.43% ± 0.66% by using triphenyl phosphate as a reference, and 98.45% ± 0.44% by using phosphonoacetic acid as a reference. Additionally, the quantification of tris(2-chloroethyl) phosphate on the basis of ¹H qNMR yielded purities of 98.41% ± 0.53% by using triphenyl phosphate as a reference, and 98.88% ± 0.52% by using phosphonoacetic acid as a reference. All these results derived from different nuclei and different traceability chains are summarized in Figure 5, and the consistency of the data is demonstrated by the overlap of their expanded measurement uncertainties. A potential combination of triphenyl phosphate and phosphonoacetic acid for quantification within the same NMR tube was not possible, due to their differing solubilities (polar and non-polar). It is important to note that the experiments were independent of each other; also, different measurement systems had to be tested in each case to find a suitable reference standard, including selecting the appropriate deuterated solvent and elaborating the appropriate acquisition parameters, such as relaxation times.

Measurement Uncertainty

The quantification of the purity in the experiments shown is based on the so-called internal standard method, where the analyte signal is directly compared to an internal reference signal. This approach can be applied not only for ¹H, but also for ³¹P signals. The CRM content is finally expressed as percent mass fraction. It should be noted that even minor uncertainty contributions from air buoyancy correction were taken into account. Therefore, climate data were recorded during each weighing step. The uncertainty calculation is based on well-established guidelines. The combined standard uncertainty is determined by statistical as well as systematic contributions; the statistical contribution arises from the repeatability of weighing and signal integration. Various systematic contributions, such as the air buoyancy correction, balance parameters, molecular masses, and the purity of the reference (expressed as a mass fraction), were taken into account. Phase correction and the integration of the signals were done manually and may differ slightly with the operator. This individual influence is considered in the overall uncertainty budget as “individual integration contribution”. ³¹P has a relative receptivity of 0.0665, which means it is less sensitive than ¹H (receptivity of 1.00), thereby leading to a lower signal-to-noise ratio. This can be compensated for by a higher analyte concentration or a higher number of scans, but it has to be considered that higher analyte concentrations can lead to reduced solubility and therefore higher viscosity or broadening of the signals.

Conclusions

The certification concept for ³¹P qNMR CRM described has successfully shown how traceability to an SI unit can be established by qNMR using different nuclei. Within this concept, it was proven that purity values of a single material (phosphonoacetic acid (96708)) using ¹H or ³¹P measurements are fully consistent with each other. Taking advantage of this concept, the content of triphenyl phosphate (05498) was determined by ¹H qNMR, but the substance with its purity value was subsequently used as ³¹P qNMR CRM. In an additional experiment, the purity of an exemplary analyte, tris(2-chloroethyl) phosphate, was measured following different traceability chains and solvent systems. Since the results are comparable within the range of their measurement uncertainties, the robustness of the certification concept is demonstrated. A possible application for the usage of these ³¹P qNMR CRMs is given by the measurements of tris(2-chloroethyl) phosphate, a sample analyte. The work described in this article represents an important step towards a successful method validation for ³¹P qNMR measurements.