M. Hämmerle, et al.
AnalyticalBiochemistry601(2020)113778
into nicotinic acid [3]. Finally, ‘nucleoside salvage pathway’ which
converts nicotinic acid riboside and nicotinamide riboside to NAD+. In
the investigation of vitamin B3 deficiency in animal populations due to
insufficient tryptophan and/or insufficient bioavailable vitamin B3 in
the diet, it is not reliable to measure the levels of NAD+/NADP+ in
blood or other tissues because these redox coenzymes are involved in
many metabolic and signalling pathways and therefore their levels
could fluctuate due to a multitude of factors and depending on the
redox state. In animal studies where the subjects are dead at the time of
sampling or a liver biopsy is possible, it is most reliable to measure total
vitamin B3 levels in the liver since the de novo biosynthesis of nicotinic
acid (and NAD+ thereafter) occurs in the liver.
mL, then 250 μL aliquots were added to 1.00 mL of methanol and
500 μL of H2O and pH was adjusted to 5–7 using NaOH with Tashiro's
indicator. Methyl nicotinate was then extracted with 1.00 mL of hexane
(≥97.0% CH3(CH2)4CH3, Sigma/Aldrich) or cyclohexane (C6H12
,
Sigma/Aldrich) or heptane (≥99.0% CH3(CH2)5CH3, Sigma/Aldrich)
or chloroform (99.0–99.4% CHCl3, Sigma/Aldrich) by vortexing for
3 min, and transferred into GC vial for analysis (1.00 μL injection).
Extractions were performed in four replicates followed by two GC runs
of each replicate sample. For the best extractant, chloroform, the ex-
tractions were then repeated six more times for the determination of
average extraction efficiency. Each replicate sample was run twice on
the GC.
Several analytical methods for detection and quantification of ni-
cotinic acid and/or nicotinamide are available. Microbiological
methods are laborious and lack precision while colorimetric methods
lack specificity and sensitivity [9–11]. More recently, methods based on
liquid chromatography (LC, HPLC) are used; however, good separation
of underivatized nicotinic acid/nicotinamide from other bioanalytes is
needed if the detection is based on ultraviolet (UV) absorption
[9,11–13]. For more sensitive and selective fluorescence-based detec-
tion/quantification, post-LC-column derivatization is needed [9,10,13].
LC-based methods can determine the sum of all forms of vitamin B3 if a
pre-column acid or base treatment of sample is included. Under
strongly acidic or basic aqueous conditions, free nicotinamide and ni-
cotinamide moiety of NAD+/NADP+ are converted to free nicotinic
acid and therefore only detection/quantification of nicotinic acid is
required (9,12). LC with mass spectrometric detection (LC-MS) cir-
cumvents the UV- and fluorescence-based difficulties and can poten-
tially determine all three forms of vitamin B3 individually but requires
separation procedures to minimize ‘ion suppression’ and also isotope-
coded internal standards are usually needed for quantification [11,14].
Similarly, gas chromatography-mass spectrometry (GC-MS) is robust
but also usually requires isotope-coded internal standards for quanti-
This report describes a method for the quantification of the sum of
the three forms of vitamin B3 from animal liver based on gas chroma-
tography with flame ionization detection (GC-FID). Free nicotinic acid,
free nicotinamide and nicotinamide moiety of NAD+/NADP+ (and
their riboside precursors, nicotinamide riboside and nicotinic acid ri-
boside) were simultaneously derivatized as methyl nicotinate under
strongly acidic conditions in methanol. Methyl nicotinate was suitable
for GC analysis due to its chemical properties such as volatility and
solubility (see discussion). Furthermore, pooling of all vitamin B3 forms
together allowed for the determination of the total levels of vitamin B3
while simplifying the separation and detection/quantification pro-
cesses. Reliable investigation of vitamin B3 levels in animal populations
often requires affordable, simple, and fast yet sensitive and re-
producible quantitative methods for the analysis of hundreds of samples
enabling accurate statistical analysis. The method was used to quantify
vitamin B3 from three wild boar livers with good within-laboratory
reproducibility. The accuracy of the method was determined from the
recoveries of the pre-extraction spiked-in standards (each of the three
vitamin B3 forms individually and all together). The matrix effect was
assessed from the post-extraction spike-in of methyl nicotinate stan-
dard. Additionally, the identity of methyl nicotinate product from liver
was confirmed by GC interfaced with an electron ionization mass
spectrometer (GC-EIMS).
2.2. Optimization of the acid-catalyzed methyl esterification of the three
forms of vitamin B3
2.2.1. Optimum reaction conditions for acid-catalyzed methyl esterification
For the determination of ideal reaction temperature, H2SO4
(95.0–97.0%, Sigma/Aldrich) was added (final concentration of
311 mM) to a solution of 0.500 mg/mL nicotinic acid (C6H5NO2,
123.11 Da, ≥99.5%, Sigma/Aldrich), or 0.500 mg/mL nicotinamide
(C6H6N2O, 122.12 Da, ≥99.5%, Sigma/Aldrich), or 40.0 μg/mL nico-
tinamide adenine dinucleotide (NAD+, C21H27N7O14P2, 663.43 Da,
Sigma/Aldrich), or 40.0 μg/mL nicotinamide adenine dinucleotide
phosphate (NADP+, C21H28N7O17P3, 743.41 Da, Sigma/Aldrich) in
methanol (final reaction volume = 1272 μL). Reaction mixtures in
sealed vessels were heated in a water bath for 30 min at either 60 °C,
80 °C, or 100 °C. Samples were then cooled, 500 μL of H2O added, and
pH was adjusted to 5–7 using NaOH with Tashiro's indicator. Methyl
nicotinate was then extracted with 1.00 mL of chloroform by vortexing
for 3 min, and transferred into GC vial for analysis (2.00 μL injection).
For the determination of ideal reaction time, H2SO4 was added (final
concentration of 311 mM) to a solution of 0.100, 0.500, and 1.00 mg/
mL nicotinic acid, or 0.100, 0.500, and 1.00 mg/mL nicotinamide, or
40.0, 80.0, and 160 μg/mL NAD+ in methanol (final reaction
volume = 1272 μL). Reaction mixtures in sealed vessels were heated in
a water bath at 100 °C for 15, 30, 45, 60, 75, 90 min (for nicotinic acid
or nicotinamide reactant), or at 100 °C for 5, 10, 20, 30, 40 min (for
NAD+ reactant). Samples were then cooled, 500 μL of H2O added, and
pH was adjusted to 5–7 as above. Methyl nicotinate was extracted with
1.00 mL of chloroform by vortexing for 3 min, and transferred into GC
vial for analysis (1.00 μL injection). In each experiment and for each
experimental condition, samples were processed at least in triplicates
followed by two GC runs of each replicate sample.
2.2.2. Within-laboratory reproducibility of the acid-catalyzed methyl
esterification and chloroform extraction of methyl nicotinate
The within-laboratory reproducibility of the optimized procedure
was determined for the methyl esterification and chloroform extraction
of all three reactants together in six replicates. H2SO4 was added (final
concentration of 311 mM) to a solution of 0.500 mg/mL nicotinic acid
and 0.500 mg/mL nicotinamide and 80.0 μg/mL NAD+ in methanol
(final reaction volume = 1272 μL). Reaction mixtures in sealed vessels
were heated in a water bath at 100 °C for 90 min. Samples were then
cooled, 500 μL of H2O added, and pH was adjusted to 5–7 as above.
Methyl nicotinate was then extracted with 1.00 mL of chloroform by
vortexing for 3 min, and transferred into GC vial for analysis (1.00 μL
injection). Each replicate sample was run twice on the GC.
2. Experimental
2.3. Stability of methyl nicotinate in methanol/sulfuric acid
2.1. Optimization of the extraction procedure for methyl nicotinate
H2SO4 was added (final concentration of 311 mM) to a solution of
0.200 mg/mL methyl nicotinate standard in methanol (final
volume = 1272 μL). Solution mixtures in sealed vessels were heated in
a water bath at 100 °C for 15, 30, 45, 60, 75, 90 min. Samples were then
cooled, 500 μL of H2O added, and pH was adjusted to 5–7 as above.
The methanol:H2O:extraction solvent volume ratios were optimized
for maximum extraction efficiency (%). For the optimized extraction,
methyl nicotinate standard (C7H7NO2, 137.14 Da, 99%, Sigma/Aldrich)
was initially dissolved in methanol (≥99% CH3OH, Roth) at 1.00 mg/
2