CLIN. CHEM. 22/8, 1322-1329 (1976)

1322 CLINICALCHEMISTRY,Vol. 22, No. 8, 1976

Purification and Analysis of the Purity of NADH

Sam A. Margolls, Barbara F. Howell, and Robert Schaffer

We developed an analytical reverse-phase high-perfor-mance liquid chromatographic procedure for rapid as-sessment of the purity of NADH. The method completelyseparates adenosine monophosphate and adenosine di-phosphoribose from NADH. By use of this analyticaltechnique we found that preparative chromatography onDEAE-cellulose gives NADH that is free from adeninenucleotides as well as other impurities that commonly arepresent in NADH. The absorbance ratio at 260 and 340 nmof the purified NADH in 1.8 mmol/liter ammonium car-bonate is 2.261 ± 0.002 (±1 SD).

AdditIonal Keyphras.s: reverse-phase hip-performance liquidchromatography #{149}preparative chromatography on DEAE-cellulose #{149}ADP #{149}ADP-ribose #{149}NADH stability #{149}impuritiesin NADH preparations #{149}spectrophotometricproperties of pure NADH

Preparations of reduced nicotinamide adenine di-nucleotide (NADH) invariably seem to contain im-purities, some of which inhibit dehydrogenase activity(1-3). Some of these contaminants can be detected byuse of either thin-layer (4) or column chromatography(2); however, the relative proportions of impurities canbe more easily evaluated by column-chromatographicmethods, especially with use of ultraviolet-sensitivecontinuous-flow detectors. The column methods thathave been found effective involve ion-exchange chro-matography (usually with DEAE-cellulose1 under al-kaline conditions), but they are unable to separate allof the adenine nucleotides satisfactorily from NADH(1, 2, 5-7). Ion-exchange methods at acidic pH (8) orthat involve use of multivalent anions (9-11) are inap-propriate because NADH is unstable under such con-ditions (13-15).

Bioorganic Standards Section, Analytical Chemistry Division,National Bureau of Standards, Washington, D. C. 20234.

Nonstandard abbreviations used: ADP-ribose, adenosine 5’-

diphosphoribose; DEAE-cellulose, diethylaminoethyl-cellulose;HPLC, high-performance liquid chromatography; , molar absorp-tivity; NBS, National Bureau of Standards; and SRM, StandardReference Material.

Identification of any commercial product does not imply recom-mendation or endorsement by the National Bureau of Standards, nordoes it imply that the material or equipment identified is necessarilythe best available for the purpose.

Received Mar. 10, 1976; accepted May 13, 1976.

We report here a rapid quantitative method forNADH analysis by high-performance liquid chroma-tography (HPLC), specifically, reverse-phase liquidchromatography. The reverse-phase chromatographycomplements the separation achievable on DEAE-cel-lulose, in that it resolves impurities in NADH that arenot completely separable on DEAE-cellulose. With ourmethod we have analyzed NADH that we had purifiedon DEAE-cellulose under improved chromatographicconditions and also some representative commercialsamples of NADH. Some of the properties of highlypurified NADH are reported.

Materials and MethodsReagents

NADH was purchased from Sigma Chemical Co., St.Louis, Mo. 63178; P-L Biochemicals, Inc., Milwaukee,Wis. 5305; Worthington Biochemical Corp., Freehold,N. J. 07728; Boehringer Mannheim Corp., New York,N. Y. 10017; and Calbiochem, La Jolla, Calif. 92037.Adenosine, AMP, ADP, ATP, and ADP-ribose wereobtained from Sigma Chemical Co.

NAD was obtained from Sigma Chemical Co. andfrom P-L Biochemicals, Inc.

The identity of each adenine nucleotide was con-firmed by thin-layer chromatography of about 50-tgsamples in the following systems:

Polyethyleneimine-cellulose (Brinkman Instruments,Inc., Westbury, N. Y. 11590) with 1.0 mol/liter LiCl asdeveloping solvent (4).

Cellulose with 7/3 (by vol) ethanol/ammonium ace-tate (1.0 mol/liter, pH 7.5) as developing solvent (12).

Cellulose with 66/33/1 (by vol) isobutyric acid!water/concentrated ammonium hydroxide as devel-oping solvent (22).

DEAE-cellulose, Whatman DE52, was purchasedfrom W&R Baiston Ltd., Maidstone, Kent, England.

Apparatus

The HPLC system was composed of a solvent deliverysystem assembled from a Milton-Roy minipump (Model396; Milton-Roy Co., Riviera Beach, Fla. 33404); a glasssolvent reservoir that could be heated to deaerateaqueous solvents; stainless-steel tubing, 0.76 mm (.030

4

C

B

A

24

CLINICALCHEMISTRY,Vol. 22, No. 8, 1976 1323

in.) i.d., to conduct the solvent to a high-pressure sampleinjection valve [Model CV-6-U, No. PaC-20, pressurelimit of 48.3 MPa (7000 psig); Valco Inst. Co., Houston,Tex. 77024]; a 10-cm length of stainless-steel tubing, to

connect the injector valve to the “iBondapak C18”column (No. 27324; Waters Assoc. Inc., Milford, Mass.01757); and a 254-nm ultraviolet monitor (Model No.1205; Laboratory Data Control, Riviera Beach, Fla.33404) with a ServoRiter II recorder (Texas Instru-ments, Inc., Houston, Tex. 77006). At wavelengths otherthan 254 nm, we used a variable wavelength monitor(Model SF770; Schoeffel Inst. Co., Westwood, N. J.07675).

Methods

Absorbances were measured with a Cary 14 spectro-photometer, which was periodically calibrated forwavelength accuracy against an NBS holmium oxidefilter and against the deuterium-lamp emission spec-trum. Its absorbance scale was periodically calibratedwith a set of NBS glass absorbance filters (SRM 930b)and solutions of potassium dichromate (SRM 136c) inaqueous 50 mmol/liter sulfuric acid. The absorbancesat 260 mn were measured with a cuvet in a thermostatedholderat 25#{176}C,and the measured values were correctedfor solvent blank.

The prepacked iBondapak C18 columns were equil-ibrated with sodium potassium hydrogen phosphatebuffer (20 mmol/liter, pH 7.05) at room temperature.The system was operated at 17.25 MPa (2500 psi), buton prolonged use flow rates gradually decreased from0.8 to 0.55 mI/mm. Sample volumes were defined by useof a 200-al sampling loop. Samples were dissolved in 1.8mmol/liter ammonium bicarbonate, pH 10.2, exceptwhere other buffers are specifically indicated. The chartspeed of the recorder was 0.33 cm/mm. Absorbanceswere detected at 254, 285, and 340 nm. The sensitivityof the detectors was attenuated for the major peak asindicated in the figures. Peak areas were measured witha planimeter.

DEAE-cellulose was conditioned by suspending it for0.5 h in aqueous 0.5 mol/liter NaOH and 0.5 mol/literNaC1 and then washing it twice with equal volumes ofdistilled water. As a second step it was suspended for 0.5h in 0.5 mol/liter HC1 and 0.5 mol/liter NaC1 and washedtwice with equal volumes of distilled water. The firststep was repeated, and then the DEAE-cellulose waswashed with distified water until the pH of the washingswas neutral. Depending on the planned use, it was nextsuspended for 16 h in 0.5 mol/liter ammonium bicar-bonate at either pH 8.2 or pH 10.2, before being packedinto a column. For experiments done by following themethod as described by Strandjord and Clayson (1), theDEAE-cellulose in the packed column was equilibratedat 4#{176}Cwith 10 mmol/liter ammonium bicarbonate, pH8.2, and the NADH, in 10 mmol/liter ammonium bi-carbonate, was added. In the chromatography we usedan ammonium bicarbonate gradient from 10 to 110mmol/liter at pH 8.2. The NADH began to emerge fromthe column as the ammonium bicarbonate concentra-

TIME (mm)

Fig. 1. Reverse-phase HPLC analysis of a relatively pure com-mercial sample of NADH, with detection at three wavelengths:254, 285, and 340 nmNADH from source 5, lot 2, was dissolved in 10 mmol/Ilter ammonium bicar-bonate, pH 8.2, at a concentration of about 0.5 mmol/ilter. The absortiance scalewas at 0.02 A for 254 nm (Curve A) and at 0.01 A for 285 ran and 340 nm (CurvesC and B). and attenuated as indicated by the values at the breaks in the chro-matograms. The Asss/Ao ratio was 2.36 1

tion got near the higher value. For our modification oftheir method, the packed column was equilibrated at4#{176}Cwith 1.8 mmol/liter ammonium bicarbonate bufferat pH 10.2. The NADH (40 mg in 0.5 ml of this buffer)was applied to the column and chromatographed withthe same buffer. Furthermore, the buffers were purgedwith nitrogen and kept under nitrogen, and fractionswere collected and stored under nitrogen at 4 #{176}Cin thedark.

ResultsHPLC of a relatively pure commercial NADH is il-

lustrated in Figure 1. Curve A represents the chro-matogram detected at 254 nm, curve B at 340 nm, andcurve C at 285 nm. Ten peaks are resolved in additionto the major peak, identified as peak 11, which absorbsat both 254 and 340 nm and is due to NADH. Peaks 2,3, and 10 show the retention times for AMP, ADP-ri-bose, and NAD, respectively (see Figure 2). Some ofthese peaks are also found on chromatography ofNAD+. Thus, Figure 2A shows the presence of smallproportions of AMP (peak 2), ADP-ribose (peak 3), andpeaks 6 and 9 plus still smaller amounts of peaks 1 and7, but there is no peak that by 254-nm or 340-nm de-tection corresponds to NADH. Products that are dueto acid degradation (14, 15) of NADH are also knownto occur on prolonged exposure (14) of NADH tophosphate buffer at pH 7. A chromatogram obtained

t

4

4

4

\---

TIME (mu,)

24 48

TIME (mun(

with NADH under the latter conditions is shown inFigure 3. Comparison of Figures 1 and 3 illustrates theextent to which degradation products are formed onstorage of NADH inS mmol/llter phosphate for 25 days.These products include peaks 5,6,8, and 10. The acid-degradation products of NADH are known to exhibitan absorption maximum (14) between 275 and 285 nm,

and the chromatogram depicting measurement at 285nm (see Figure 1C) confirms that compounds 5 and 8have the increased absorbance at 285 nm characteristicof the acid-degradation products.

Quantitation of Known Compounds

The HPLC method for separation of the adeninenucleotides from NADH can provide the basis for theirquantitation. Figure 4 illustrates the linear relationships

Table 1. Summary of the HPLCCommercIal samples

Peak Areas of Samples of NADHDEAE-cellulose chromatography a

23

5a5b

6

7

8

58

27

11

19

171534 b

14

111

1020

9550

29 ‘

111

08

2413

17”

Commercial NAD+

22

13

2726

13

1019b

31

48

06

1

5

10b

0

00

0

0

0

19

15

00

0

1

0

Peak no. 1 2 3 4 5 Fract. 20, Day 4 Fract. 1254 nm

2 3 1 4 1 3

9 19.210 26311 433112 15

340 nm1 11 6 7 3 7 0 0 04 0 0 0 0 4 0 0 0

10(NAD) 12 16 4 1 0 0 0 511(NADH) 1973 1933 2250 1773 2540 454 101 0

179

41600

125

4872

0

1

1820

0

15

30

115

3851

0

98

5528

0

4

972

0

2150

1259

0

0

#{149}Fractions are from the experiment illustrated in Figure 7. b Sum area of peaks 8 and 9.

0

1324 CLINICALCHEMISTRY,Vol. 22, No. 8, 1976

Fig. 2. Reverse-phase HPLC analysis of commercial AMP,ADP-ribose, and NAD, showing their retention timesEach nucleotide was dissolved in 5 mmoi/Ifter phosphate buffer, p117.0. at aconcentration of about 0.2 mmol/llter. The absorbance scale was 0.02 A for254 nm and 0.01 A for 340 nm, and attenuated as indicated by the value at thebreaks in the chromatograms. Curve A is for NAD. curve B for AI*’, and curveC for ADP-ribose, each detected at 254 nm. CurveD Is for NAD detected at340 nm

Fig. 3. Reverse-phase HPLCanalysis of NADH(thesame as usedfor Figure 1) dissolved in 5 mmol/llter phosphate buffer, pH 7.0,and stored for 25 daysThe concentration of NADHwas about 0.5 mmol/Ilter. The absorbance scalewas setat .02 A for 254-nmdetection(csve A)andato.01 A for 340-nmde-tection (curve B), and attenuated as indicated by the values at the breaks in thechromatograms

tooI

504

ISO

f 100

4

50

I50

I00

4

50

20 40 100 20

Ui0z.4

0Co0.4

60 80

FRACTION NO.

Fig. 6. Chromatogram of NADH (5 mg) on DEAE-cellulose, ac-cording to ref. 1Curve A represents A,o; curve B, A; and curve C,

1.5 uuu J 38

2.34#{176}

II

N/ j__.<8 2.30

N/ \ 1C ‘-.

I #{149}-#{149}‘.. #{149}d #{149}

I I I I

.5 1.0 .5 2.0

40X

24 48

C

IC

C

IC

24 48TIME unnI

24 48

C

24

TIME (unnI

24 48

58

between peak areas and quantities of AMP, ADP-ri-bose, and NADH. Because none of these nucleotides issupplied commercially as a highly pure substance (seeFigure 2), and each may contain an uncertain propor-tion of water, the concentrations of solutions prepareddirectly from weighed quantities of such preparationscannot be assumed to be accurate; however, each can beused to assess the relative amounts of the compound indifferent preparations of NADH from the areas of thepeaks (see Figure 5 and Table 1).

Preparation of Pure NADH

Ion-exchange chromatography on DEAE-celluloseaccording to the methods of Strandjord and Clayson (1)and of Silverstein (6), typically separates NAD com-pletely, and ADP-ribose and AMP partly, from NADH(see Figure 6). However, by use of their methods, theA2 to A340 absorbance ratio of the NADH fractionsdoes not become constant, which indicates contami-nation by differing proportions of impurities. We havefound, by modifying their separation conditions in thefollowing ways, that NADH can be obtained that givesa constant A2,0 to A340 ratio:

24

TIME(oun)48

CLINICALCHEMISTRY,Vol. 22, No. 8, 1976 1325

zuI

NADH

5 .0 1.5 2.0m9/mI

Z

ADP RIBOSE

.5 .0 .5 2.0ng/uI

“““u

AMP

I

n/nuI

Fig. 4. Calibration curves for AMP, ADP-ribose, and NADHanalyses of peak heights by reverse-phase HPLC, showing thelinear relationship between peak area and concentrationPeak areas were measured with a planimeter. The nucleotide samples weretaken from separate solutions from which 200-SI aliquots were chromatographed

SC

24 48TIME (ur.nI

L=jJ

48

48

FIg. 5. Reverse-phase HPLC analysisof NADH from differentcommercial sourcesThe concentration of NADHfor each chromatogram was about 0.6 mmol/llter.A represents the chromatograms of NADHfrom source 1, B from source 2, Cfrom source 3, and D from source 4. The AIA ratios for each sample were,respectively, 2.620,2.542,2.430, and 2.388. The u,erchromatogram in eachset represents A; the lower, A2. The absorbance scale was 0.02 A for 254nn, and 0.01 A for 340 nm, and was attenuated as indicated by the values at thebreaks in the chromatograms

UJ0 1.0

0U).4

0.5

2.26

20 40 60FRACTION .

Fig. 7. 260- and 340-nm absorbances, as measured with aspectrophotometer,and ratios of sequentiai NADHfractions fromthe DEAE-cellulose chromatography experiment Illustrated inthe insetCurve A represents A; curve B, A;and curve C, Inthe ksef, curveA represents A2M and curve B, A,.0

7

9

12

17

Mean AaJJIA34.o

2.263

2.263

2.262

2.261

#{149}NADH from sourceS. Two different lots were used.

8Number of consecutive fractions from the NADH peak used to obtain the

mean.

-

C

0

.4

IICd

‘4

24 48

‘

TIME (mm)

Fig.8. Reverse-phase HPLC analysis of NADH fractIons afterprior chromatography on DEAE-cellulose In the experiment Il-lustrated in Figure 7The fractIon numbering is that used in FIgure 7. Curve A represents the ctwo-matogram of fraction 12; curve B is for fraction 16; curve C Is for fraction 20;curve DIs for fraction 25; curve E Is for fraction 28; and curve F is for fraction32. The upper chromatogram represents A; the lower. ASM. The numbersat the breaks in the ctwomatogams represent the attenuation factors from amaximum sensltIvfty of 0.02 A at 254 nm and 0.01 A at 340 nm

1326 CLINICALCHEMISTRY.Vol. 22, No. 8, 1976

(a) The eluting buffer, ammonium bicarbonate, ispurged with nitrogen before use, and the fractions arecollected and stored under nitrogen; (b) the entire ex-periment is performed in the dark at 4 #{176}C;and (c) theelution is done with the ammonium bicarbonate bufferkept at a concentration of 1.8 mmol/liter and at a pH of10.2. The result is illustrated in Figure 7. Four impuri-ties are completely resolved in the early fractions, andthe adenine nucleotides appear on the leading edge ofthe NADH peak. Because of their presence, the A200 to

A340 ratios of successive NADH fractions exceeded 2.261through fraction 12, but thereafter the ratio becameconstant at 2.261 ± 0.002 (1 SD, see Table 2). Theinter-experiment reproducibility of this ratio for severalexperiments is illustrated in Table 3, in which two dif-ferent lots of NADH from source 5 were chromato-graphed. These values of the absorbance ratio are lowerthan previously cited values (16, 17).

HPLC Reverse-Phase ChromatographicEvaluation of Purity and Stability of NADHSolutions Purified on DEAE-Cellulose

The purity of these latter fractions of NADH thateluted from the DEAE-cellulose was shown by re-verse-phase HPLC (see Figure 8). Only peak 10, thepeak area of which was less than 0.5% that of the NADHpeak, could be detected when these NADH fractionswere examined one day after the DEAE-cellulose

Table 2. A260/A0 Ratios Calculated forFractions of an NADH Peak from DEAE-Cellulose

ChromatographyFractIon no. A260 A340 A26OJASao

1 .518 .193 2.684

4 .950 .399 2.38 1

8 1.496 .649 2.305

12 1.610 .712 2.261

16 1.580 .699 2.260

20 1.501 .663 2.264

25 1.415 .626 2.260

28 1.323 .585 2.262

32 1.247 .552 2.25936 1.156 .511 2.262

40 1.064 .471 2.259

42 1.028 .455 2.25944 0.982 .434 2.263

48 0.89 1 .394 2.26 1

52 0.809 .358 2.260

56 0.726 .321 2.262

60 0.646 .286 2.259

64 0.577 .255 2.263

68 0.506 .224 2.259

72 0.446 .197 2.264

Fractions 12-72 = 2.26 1

SD = ±0.002n = 17

Table 3. RepeatabIlity (Four Experiments) of theA260/A340 of NADHC

±1 SD

1.4 X iO

2.0 X io-

2.1 X i0-32.0 X iO-

chromatography was completed, and the proportion ofthis impurity peak had hardly increased by day 4[compare fraction 20 on day 1 (Figure 9) and day 4(Figure 8)j. In the impure initial NADH fractions fromthe DEAE-cellulose chromatography the impurities areAMP and ADP-ribose, as indicated in the chromato-gram of fraction 1 of Figure 9. These compounds areabsent in the later fractions that exhibited the constantA2IA3.

Table 4 provides data on the spectrophotometricstability of the NADH eluted from the DEAE-cellulosecolumns. These measurements were made on (a) a poolof NADH fractions that exhibited the constant ab-sorbance ratio and (b) separate NADH fractions ofsimilar quality from another experiment. The data showthat, when stored under the conditions we used for theDEAE-cellulose chromatography, the NADH under-went little change over a period of several days.

0

C

12

357

Table 4. StabIlity of NADH in 1.8 mmol/llterAmmonlum Bicarbonate, pH 10.2, Stored at 4 #{176}C

in the DarkSample age (days) A2as A34o A25r.i/A340

Experiment1 (pooled sample)1.015 .449 2.263

1.022 .450 2.271

1.005 .444 2.264

1.019 .450 2.264

1.023 .447 2.288

Experiment 2 (selected fractions)2(Fractionl2) 1.610 .712 2.261

4(Fractionl2) 1.610 .712 2.261

2 (Fraction 16) 1.580 .699 2.260

4 (Fraction16) 1.589 .702 2.264

1 (FractIon 20) 1.501 .633 2.264

a After elution from DEAE-celluiose column.

B

A

24 48TIME (mint

4

4

4

Fig. 9. ComparIson by reverse-phase HPLC analysis of crudeNADHand column purlfled NADHfractions having hIgh and lowA260/AA, thromatograms recorded at 254 nm; B, ctwomatograms recorded at 340 nm.Curve A is the ctwomatogram from If IC of fractIon 1 from the OEAE-cellulosefractionation depicted in Figure 7. Similarly, curve B corresponds to fraction20. Curve C represents the ctwomatogram of the NADHfrom sourceS, lot 2,before fractionation on OEAE-cellulose. Curve D represents the clwomatogramof the 1.8 mmol/Iiter ammonium carbonate buffer, pH 10.2. The absorbancescale was .02 A, for 254 nm and 0.01 A for 285 and 340 nm; and ftwasatten-uated as Indicated by the values at the breaks in the chromatograms

D

Commonly accepted values Observed values

B

NADH, NBS DEAE-

Ratio P-L (17) SIgma (18) source 5 chromatography

A260/ 2.32 2.3 2.322 2.26 1

A340 ± 0.002

A280/ 0.24 0.23 0.250 0.234

A260A260/ 4.00 4.27

A280A260/ 9.34 10.35

A290 ± 0.18

A290/ .107 0.093

A260A250/ 0.80 0.81 0.796 0.794

A260

24 48TIME (minI

CLINICALCHEMISTRY,Vol. 22. No. 8, 1976 1327

Table 5. Absorbance Ratios of NADH and TheirValues

Spectrophotometric Properties of Pure NADH

Absorbance ratios at specific wavelengths are com-monly measured to assess NADH purity. These ratiosreflect degradation products that form from NADHunder acidic conditions, absorbing in the 275- to 285-nmwavelength region (14, 17, 18), and other impuritiescommonly found in NADH, absorbing only in the regionof 258 to 260 nm. Table 5 lists the usually measuredratios-i.e., the values that have been commonly con-sidered to reflect optimum NADH-the values wefound for the best commercially obtained NADH wehave identified, and our values for the latter NADHafter it had undergone purification by DEAE-cellulosechromatography.

260 and330-370

1,4, 10

260 and 290 5,7,8, 6 (‘?)

260 2, 3, 9, 10,12

1328 CLINICALCHEMISTRY,Vol.22,No.8,1976

In the ultraviolet spectrum of purified NADH thatexhibits the low, constant A260 1A340 ratio, maximumabsorbances were at 259.4 and 339.5 nm, and the ab-sorbance minimum between these peaks was at 290.0nm.

DiscussionThe use of ion-exchange for the chromatography of

NADH is well documented (1, 5,6, 7), but with none ofthese methods can one completely remove the knowndehydrogenase inhibitor, ADP-ribose, from the NADH.

Initially, ADP-ribose was detected as an NADHcontaminant by Dalziel (2) by using paper chromatog-raphy. Silverstein (6) attempted to separate an“ADP-ribose-like” contaminant from NADH by chro-matography on DEAE-cellulose. This material tendedto concentrate in the first part of the NADH peak, butas indicated by the A0/A ratio, which decreased andthen increased, pure NADH was never obtained. Zie-

genhorn et al. (20) recently reported that the middlesegment of the NADH peak obtained by chromatogra-phy on “QAE-Sephadex” exhibited a constant A260 toA340 ratio, but rechromatography in the same systemindicated that the NADH peak still contained signifi-cant ADP-ribose inthe first four fractions. The ratio forthe next seven fractions was 2.265 ± 0.012 (1SD). Thisvariabilitycould be due to the continuing presence of1% of ADP-ribose or other 260-nm absorbing materialsamong the latter fractions.

The chromatographic conditions for purifying NADHare severely restricted by the reactivity of NADH (14),by its instability at acid pH (19), and by the insolubilityof this highly polar compound in various commonlyused chromatographic solvents. Reverse-phase HPLCcan be performed under conditions conforming to the

stability requirements for NADH, and the method hasthe additional advantage of being rapid. By applyingreverse-phase HPLC to a “good” commercial NADH,10 impurity peaks can be separated. These peaks are notartifacts of chromatography, because (a) they have beenobserved in 10-fold different proportions in the com-mercial samples studied (see Figure 5), (b) their dis-tribution is independent of the pH of the dissolvingbuffer, and (c) they may be present in trace amounts orbe completely absent in preparations of highly purifiedNADH (see Figure 9).

The HPLC method is particularly applicable toanalysis of small or dilute samples, and therefore small,relatively dilute fractions of NADH eluted from aDEAE-cellulose column can be evaluated. The resultsof such an evaluation are illustrated in Figure 9, whichshows that the initial fractions of the NADH obtainedby DEAE-cellulose chromatography contain bothADP-ribose and AMP, whereas the later fractions,which exhibit the constant A260 to A340 ratio, do notcontain these nucleotides. These fractions show only asingle impurity peak, which elutes like NAD. Its areaby 254-nm detection corresponds to less than 0.5% ofthe area of the NADH peak.

With the aid of standards, some of the peaks in the

Table 6. ClassIfication of impurities Detected InNADH by Reverse-phase HPLC

Absorption Impurity peak

Group Source of Impurity peaks (nm) no.

1 NAD adducts (3,?)NAD alkaline

degradation (19)2 NADH aciddegradation

(14, 15), and NMN3 Other nucleotides,

NAD, AMP, ADP-

ribose

chromatograms of commercial NADH can be identified.Others can be classified according to their spectralproperties. The impurities may be categorized accordingto their absorption range as (a) NAD adducts andNAD alkaline degradation products, (b) NADHacid-degradation products and NMN, and (c) othernucleotides, as summarized in Table 6. From the use ofstandards, it is evident that peak 2 is AMP, peak 3 isADP-ribose, and peak 10 contains NAD (see Figures1 and 5). Peak 11, the major component, is NADH.Peaks 5,6, and 8 correspond to products that form fromNADH in the presence of phosphate anion, and they areclassified as group 2 (Table 6).

Peaks 1 and 4 and a part of peak 10 have not beenidentified; however, they absorb at 340 nm, which ischaracteristic both of NAD adducts and of productsof alkaline degradation of NAD. Although NADmigrates as Peak 10, a component in this peak also ab-sorbs at 340 nm. This is indicated by the fact that therelative areas of peak 10 measured at 340 nm do not varyconsistently with those measured at 254 nm (comparethe values for samples 1 and 2 and for the NAD inTable 1).

The reverse-phase HPLC method provides a meansfor critically evaluating the purity of samples of NADH.From the HPLC data in Table 1 and Figure 5, sample5 is the most nearly pure of the commercial samples ofNADH we examined. This sample contained the lowestproportion of NAD, but its content of AMP andADP-ribose was significant. This sample also showedminimal proportions of the acid-degradation productsof NADH-namely, peaks 5,6, and 8. Thus the resolu-tion provided by the HPLC method also affords themeans for revealing causes for impurity formation. Forexample, we found that oxidative and acidic degrada-tion of the NADH does not occur in the ammoniumbicarbonate buffer during storage for several days in thedark at 4 #{176}Cunder nitrogen (Table 4 and Figure 9).

NADH, purified by chromatography on DEAE-cel-lulose and shown to be essentially pure by HPLC, ex-hibits spectrophotometric properties that differsomewhat from those commonly recognized for calcu-lating absorbance ratios as indices of NADH purity (16,17). In ammonium bicarbonate at pH 10.2, the purifiedNADH has absorbance maxima at 259.4 nm and 339.5

CLINICALCHEMISTRY,Vol. 22,No.8,1976 1329

nm, and an absorbance minimum at 290.0 nm, and theA2SA0 was found to be 2.261±0.002(1 SD, see Table3). This value agrees with the value reported by Zie-genhorn et al. (2.265 ± 0.012) (20), but is more precise.Table 5 summarizes the spectrophotometric ratios. Asan index of purity we suggest that A260/A29,0 be substi-tuted for the A2601A280 ratio, because use of the ab-sorbance minimum of NADH should provide a morerational basis from which to detect the acid-degradationproducts that absorb at this wavelength region, eventhough their A290 is approximately 75% of their ab-sorbance at 280 nm (14). The differences in the ratiosof our purified NADH from the other ratios shown inTable 5 are consistent with the HPLC evidence of theabsence of NAD+ and adenine nucleotides that absorblight at 260 nm but not at 340 nm, and the absence ofGroup 2 impurities as well.

The value for A260/A340 can be used to calculate themolar absorptivity at 260 nm by use of the recentlydetermined molar absorptivity at 340 nm reported byMcComb et al. (21), who also studied the effects oftemperature, ionic strength, and pH on the absorbanceat 340 nm. By extrapolation of their data for the effectof pH on the molar absorptivity (correction for tem-perature is not needed and that for ionic strength isnegligible), their value of euH for 25#{176}C,1.8 mmol/literammonium bicarbonate, and pH 10.2 becomes 6.34 X

10g. On use of this value with our ratio of 2.261, thecalculated e#{248}?ADHis 14.335 X 10g. The commonly ac-cepted value (16) is 14.4 X 10g.

We do not advocate reliance on one ratio over theother (A2SA2.90 vs. A260/A3o). Instead,we advocate theuse of both.

We thank Ms. B. Matthews for her technical assistance. This re-search was supported by a research agreement with the National In-stitute of General Medical Sciences, NIH, USPHS.

References1. Strandjord, P. B., and Clayson, K. J., The control of inhibitoryimpurities in reduced nicotinamide adenine dinucleotide in lactatedehydrogenase assays. J. Lab. Gun. Med. 67, 144 (1966).2. Dalziel, K., Some observations on the preparation and propertiesof dihydronicotinamide-adenine dinucleotide. Biochem. J. 84, 240(1962).

3. Everse, J., and Kaplan, N. 0., Lactate dehydrogenases: Structureand function. In Advances in Enzymology, 37, A. Meister, Ed. In-terscience, John Wiley, N. Y., 1973, pp 61-133.4. Randerath, K., and Randerath, B., Thin layer separation methodsfor nucleic acid derivatives. In Methods in Enzymology, 12A, L.Grossman and K. Moldave, Eds. Academic Press, N. Y., 1967, pp323-347.

5. Pastore, E. J., and Friedkin, M., The chromatographic separationand recovery of reduced and oxidized pyridine nucleotides. J. Biol.Chem. 236,2314 (1961).6. Silverstein, E., Separation and purification of oxidized and reducednicotine adenine dinucleotides. Anal. Biochem. 12, 199 (1965).

7. DiSabato, G., Reactions of DPN and pyruvate. Biochim. Biophys.Acta 167,646 (1968).

8. Horvath, C., High performance ion-exchange chromatography withnarrow bore columns: Rapid analysis of nucleic acid constituents atthe subnanomole level. In Methods of Biochemical Analysis, 21, D.Glick, Ed., Interscience, New York, N. Y., 1973.9. Khym, J. X., An analytical system for rapid separation of tissuenucleotides at low pressures on conventional anion exchanges. Clin.Chem. 21, 1245 (1975).10. Rajcsanyi, P. M., Csillag, M., and Kriskovics, E., Separation ofnucleic acid constituents by column liquid chromatography. Sep.Purif. Methods 3, 167 (1974).

11. Singhal, R. P., Separation and analysis of nucleic acids and theirconstituents by ion exclusion and ion exchange column chromatog-raphy. Sep. Purif. Methods 3, 339 (1974).12. Paladini, A. C., and Leloir, L. F., Studies on uridine-diphospho-glucose. Biochem. J. 51, 426 (1952).13. Burton, R. M., and Kaplan, N. 0., The reaction of reduced pyri-dine nucleotides with acid. Arch. Biochem. Biophys. 101, 150 (1963).14. Alivisatos, S. G. A., Ungar, F., and Abraham, G. J., Spontaneousreactions of 1,3-substituted 1,4-dihydropyridines with acids in waterat neutrality. I. Kinetic analysis and mechanism of the reactions ofdihydronicotinamide-adenine dinucleotide with orthophosphates.Biochemistry 4, 2616 (1965).

15. Oppenheimer, N. J., and Kaplan, N. 0., Structure of the primaryacid rearrangement product of reduced nicotinamide adenine dinu-cleotide (NADH). Biochemistry 11,4675 (1974).16. Specifications and Criteria for Biochemical Compounds, 3rd ed.,Nat. Acad. of Sci., Washington, D. C., 1972, p 88.

17. UV Spectra of 5’-Ribonucleotides, Circular OR 18, P.L. Bio-chemicals Inc., Milwaukee, Wis., 1971, p 2.18. $-Nicotinamide Adenine Dinucleolide, Reduced Form, Certificateof Analysis, Sigma Chemical Co., St. Louis, Mo., 1975.19. Burton, R. M., and Kaplan, N. 0., The reaction of diphoapho-pyridine nucleotide and related pyridinium salts with alkali. Arch.Biochem. Biophys. 101, 139 (1963).20. Ziegenhorn, J., Senn, M., and B#{252}cher,T., Molar absorptivitiesof9-NADH and fl-NAD at 260 nm. Gun. Chem. 22, 151 (1976).

21. McComb, R. B., Bond, L. W., Burnett, R. W., etal., Determinationof the molar absorptivity of NADH. Clin. Chem. 22, 141 (1976).22. Krebs, H. A., and Hems, R., Reactions of adenosine and inosinephosphates in animal tissues. Biochim. Biophys. Acta 12, 172 (1953).