NAD+ is the common abbreviation for nicotinamide adenine dinucleotide in its oxidized form. Unlike most items in our catalog, it is not a peptide: it is a nucleotide-derived small molecule with a mass of roughly 663 g/mol. The page below serves as a chemistry and identity reference only. It covers structure, naming, UV and chromatographic behavior, likely impurities and mass spectrometry, and says nothing about what the substance does, nor does it offer any preparation, handling or dosing instructions.
The structure of NAD+: two nucleotides and a pyrophosphate bridge
NAD+ is assembled from two nucleotide halves joined through their phosphate groups by a phosphoanhydride (pyrophosphate) linkage. One half is nicotinamide mononucleotide, which is the nucleoside nicotinamide riboside carrying a 5′-phosphate. The other half is adenosine monophosphate (AMP), in which the purine base adenine is attached to a second ribose. Both sugars are D-ribose in the furanose form, and the naturally occurring compound is the β-anomer at the carbon linking ribose to the nicotinamide ring.
PubChem (CID 5892) lists the molecular formula as C21H27N7O14P2, with an average molecular mass of 663.4 g/mol and a monoisotopic mass of 663.1091. The CAS registry number usually cited for the oxidized form is 53-84-9. The positive sign refers to the quaternary pyridinium nitrogen of the nicotinamide ring; the two phosphates are ionized near neutral pH, so the molecule as a whole carries net negative charge in solution despite the “+” in its name.
NAD+, NADH and NADP+: telling the three names apart
Three closely related species share most of this skeleton, and labels sometimes blur them:
- NAD+ – the oxidized form, with an aromatic pyridinium ring (C21H27N7O14P2, about 663.4 g/mol).
- NADH – the reduced form. The nicotinamide ring has gained a hydride at its 4-position and is no longer aromatic, so the formula becomes C21H29N7O14P2 and the average mass rises to about 665.4 g/mol (PubChem CID 439153).
- NADP+ – a separate compound carrying a third phosphate on the 2′-hydroxyl of the adenosine ribose. Its formula is C21H28N7O17P3 and its average mass is about 743.4 g/mol, roughly 80 daltons above NAD+.
The 2-dalton gap between NAD+ and NADH is easy to miss on a low-resolution instrument, which is why the optical test described next remains the standard way to distinguish the two redox states.
UV absorbance of NAD+ at 260 nm and the NADH band at 340 nm
Adenine dominates the ultraviolet spectrum of NAD+, producing a maximum near 260 nm with a molar absorptivity of roughly 18,000 M−1cm−1. That band is the natural detection wavelength for chromatographic purity, and it is far more selective than the 214 nm peptide-bond region used for peptides.
Reduction of the nicotinamide ring creates a second chromophore. NADH shows an additional broad band centered at 340 nm (molar absorptivity about 6,220 M−1cm−1), while the oxidized pyridinium form is essentially transparent there. Comparing absorbance at 340 nm against 260 nm is therefore the classic, instrument-light way to reveal whether an NAD+ sample contains reduced material.
HPLC of NAD+: ion-pairing and HILIC methods
With four ribose hydroxyls, a charged pyrophosphate and a permanent cationic ring, NAD+ is extremely polar and elutes near the void volume on an ordinary C18 column run with a standard acetonitrile gradient. Two approaches solve this. Ion-pairing reverse phase adds a hydrophobic cation, commonly a tetraalkylammonium salt, or uses a phosphate-buffered, largely aqueous mobile phase so the anionic phosphates are retained. HILIC (hydrophilic interaction chromatography) works the other way around, holding polar analytes on a polar stationary phase under high organic content, and is often preferred when the eluent goes on to a mass spectrometer, since nonvolatile ion-pair reagents suppress ionization. Our guide on reading an HPLC chromatogram explains how peak area becomes a purity figure.
Degradants and impurities expected in NAD+
The weakest point of the molecule is the glycosidic bond between nicotinamide and its ribose. Hydrolysis there, which proceeds faster under alkaline conditions, releases free nicotinamide (C6H6N2O, 122.1 g/mol) and ADP-ribose (C15H23N5O14P2, 559.3 g/mol). Cleavage of the pyrophosphate instead gives the two mononucleotides, nicotinamide mononucleotide and AMP. Other species worth looking for are NADH, detected at 340 nm and by a +2 shift in mass, and the α-anomer of NAD+. The anomer has an identical formula and mass, so only chromatographic resolution separates it from the β form.
NAD+ powder is also noticeably hygroscopic. Analytically, this means water content is a real contributor to the weighed mass, and a thorough certificate reports it (for example by Karl Fischer titration) alongside HPLC purity rather than letting it hide inside the gross mass.
Mass spectrometry of NAD+ in negative and positive mode
Electrospray ionization handles NAD+ well in either polarity. In negative mode the deprotonated ion [M−H]− appears near m/z 662.10; in positive mode the protonated ion [M+H]+ appears near m/z 664.12. Negative mode is often favored because the phosphates ionize readily. Fragmentation typically yields ions associated with the adenosine phosphate portion and with nicotinamide, which helps separate NAD+ from isobaric background. The same accurate-mass logic described in our article on monoisotopic vs average mass applies: the monoisotopic value is the one to match on a high-resolution spectrum.
Why a 1000 mg NAD+ vial differs from a peptide vial
A typical peptide vial holds a few milligrams of a large molecule synthesized residue by residue, and the difference between gross powder mass and actual peptide depends on counter-ions and net peptide content; our explainer on peptide vial milligram labels covers that gap. NAD+ is a small molecule produced at gram scale, so a vial holding one gram is ordinary for this compound. At 663.4 g/mol, 1000 mg of the free acid corresponds to about 1.51 millimoles.
The declared form still matters. NAD+ is sold both as the free acid and as a sodium salt; PubChem lists the monosodium salt as C21H26N7NaO14P2 at about 685.4 g/mol. One milligram of that salt therefore contains only about 0.97 mg of NAD+ expressed as free acid, and any absorbed water lowers the figure further. When comparing products, check whether the stated mass refers to free acid or salt.
How NAD+ is supplied here
NAD+ 1000mg ships as a dry powder in a sealed vial. An independent laboratory runs reverse-phase HPLC on it, and that result is posted on the product page. No batch or lot codes are printed on the vials; instead, the color of the crimp and cap ties each vial to the test result published for it.
Frequently asked questions
Does NAD+ belong to the peptide class?
No. It contains no amino acids or amide backbone. It is a dinucleotide: nicotinamide mononucleotide and adenosine monophosphate linked by a pyrophosphate bridge.
What is the molecular formula and mass of NAD+?
C21H27N7O14P2, with an average mass of about 663.4 g/mol and a monoisotopic mass of 663.1091, according to PubChem CID 5892.
How can oxidized NAD+ be distinguished from NADH?
Measure absorbance at 340 nm. NADH absorbs strongly at that wavelength and NAD+ barely does. Mass spectrometry adds a check, since NADH is about 2 daltons heavier.
Why does NAD+ need ion-pairing or HILIC chromatography?
Its charged phosphates and many hydroxyl groups make it too polar to retain on plain C18 media, so an ion-pair reagent or a hydrophilic stationary phase is used to hold it on the column.
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