NAD+ quantification by HPLC and LC-MS: an analytical methods summary

Summary

This summary describes two published analytical methods for the quantification of nicotinamide adenine dinucleotide (NAD+) and the classic stability study that underlies their extraction steps. Yoshino and Imai (Methods in Molecular Biology, 2013) describe a reverse-phase high-performance liquid chromatography (HPLC) method for NAD+ in cells and tissues. Trammell and Brenner (Computational and Structural Biotechnology Journal, 2013) describe a targeted HPLC coupled to mass spectrometry (LC-MS) assay for NAD+ and the related nucleotides and explain why HPLC with ultraviolet detection alone is insufficient for complex samples. Lowry, Passonneau and Rock (Journal of Biological Chemistry, 1961) reported the stability of the oxidised and reduced pyridine nucleotides in acid and alkali.

Peptide

Name

NAD+ (beta-nicotinamide adenine dinucleotide, oxidised form). NAD+ is a dinucleotide coenzyme, not a peptide; it is included in this series because it is handled by the same analytical workflows as the peptide reagents.

Sequence

not applicable

CAS

53-84-9

Formula

C₂₁H₂₇N₇O₁₄P₂, approximately 663.4 g/mol

Class

Pyridine nucleotide coenzyme.

Study design

Yoshino and Imai 2013: a methods chapter describing reverse-phase HPLC for NAD+ determination in cell and tissue extracts. The chapter covers extraction of the sample under acidic conditions, separation on a reverse-phase column, ultraviolet detection, and calibration against NAD+ standards. The authors describe the method as highly quantitative and reproducible, and state that it enables analysis of changes in NAD+ levels in vitro and in vivo.

Trammell and Brenner 2013: a methods paper describing a targeted LC-MS assay for NAD+, its biosynthetic intermediates and related nucleotides. The paper discusses the technical challenges of reliable quantification, including ionisation suppression for particular analytes, and presents new separations that correct a problem in a previously published method.

Lowry, Passonneau and Rock 1961: a study of the stability of the pyridine nucleotides as a function of pH and temperature, which defines the conditions under which the oxidised and reduced forms survive extraction.

Endpoints: analyte identity and retention, limits of detection and quantification, linearity and reproducibility, as reported in each method.

Reported findings

Yoshino and Imai reported that their reverse-phase HPLC method gives accurate, reliable and reproducible NAD+ measurements in cells and tissues. The chromatographic conditions, detection wavelength, standard curve and validation data are given in the chapter and are not reproduced here.

Trammell and Brenner reported that HPLC followed by ultraviolet-visible detection does not offer the specificity and sensitivity necessary for robust quantification of NAD+ and related nucleotides in complex samples, and developed a targeted quantitative LC-MS assay. The new method was reported to incorporate new separations and to supersede a previously published method that suffered from ionisation suppression for particular analytes. The transitions, internal standards and validation values are in the full paper.

Lowry and colleagues reported the acid and alkali lability pattern of the pyridine nucleotides: the oxidised forms (NAD+ and NADP+) are stable in acid and labile in alkali, while the reduced forms (NADH and NADPH) are stable in alkali and labile in acid. This pattern is the reason NAD+ methods extract under acidic conditions and NADH methods extract under alkaline conditions.

Limitations

Method papers describe behaviour in the authors’ hands on the sample types they chose; transfer to another laboratory requires local validation. The 1961 stability data were obtained with the instruments of the time. None of the three papers reports biological outcomes, and none is summarised here for that purpose.

References

1. Yoshino J, Imai S. Methods in Molecular Biology. 2013;1077:203-215. PMID 24014409. DOI 10.1007/978-1-62703-637-5_14.
2. Trammell SA, Brenner C. Computational and Structural Biotechnology Journal. 2013;4:e201301012. PMID 24688693. DOI 10.5936/csbj.201301012.
3. Lowry OH, Passonneau JV, Rock MK. Journal of Biological Chemistry. 1961;236:2756-2759. PMID 14466980.