Anal. Chem. 1982, 5 4 ,
LITERATURE CITED Gross, M. L.; Chess, E. K.; Lyon, P. A,; Crow, F. W.; Evans, S.; Tudge, H. Inf. J. Mass Specfrom. Ion Phys., in press. McLafferty, F. W. Acc. Chem. Res. 1980, 13, 33-39. Kondrat, R. W.; Cooks, R. G. Anal. Chem. 1978, 5 0 , 81 A-92 A. Kruger, T. L.; Litton, J. F.; Kondrat, R. W.; Cooks, R. G. Anal. Chem. 1976, 48, 2113-2119. McLafferty, F. W.; Kornfieid, R.; Haddon, W. F.; Levson, K.; Sakai, I.; Bente, P. F., 111; Tsai, S.-C.; Schudemage, H. D. R. J. Am. Chem. SOC. 1973, 95, 3886-3892. Levsen, K.; McLafferty, F. W. J. Am. Chem. SOC. 1974, 96, 139- 144. Levsen, K.; Schwarz, H. Angew. Chem., Inf. Ed. Engl. 1876, 75, 509-519. Jennings, K. R. Chem Commun . 1988, 283-284. Tajima, E.; Selbi, J. Inf. J. Mass Specfrom. Ion Phys. 1989, 3 , I
299-303
299
(15) Weston, A. F.; Jennings, K. R.; Evans, S.; Elliot, R. M. Int. J . Mass
Spectrom. Ion Phys. 1976, 20, 317-327. (16) Russell, D. H.; Smith, D. H.; Warmack, R. J.; Bertram, L. K. Int. J. Mass Spectrom. Ion Phys. 1980, 35, 381-391. (17) McLaffetty, F. W.; Todd, P. J.; McGiivery, D. C.; Baidwin, M. A. J. Am. Chem. SOC. 1980, 102, 3360. (18) Harrison, A. G.; Hegedus-VaJda,J.; Middlemiss, N. E.; Zwinselman, J. J.; Nibbering, N. M. M. Proceedings of the 28th Annual Conference on Mass Spectrometry and Aiiied Topics, May 25-30, 1980, New York. (19) Zwinselman, J. J.; Nlbbering, N. M. M.; Middlemiss, N. E.; HegedusVaJda, J.; Harrison, A. G., submitted for publication in I n f . J. Mass Specfrom Ion Phys (20) Hass, J. R.; Bursey, M. M.; Kingston, D.G. I.; Tannenbaum, H. P. J. Am. Chem. SOC.1972, 9 4 , 5095.
.
.
-34.5-356 .- -- - .
Russell. D. H.; McBay, E. H.; Mueller, T. R. Am. Lab. (Fairfiekl, Conn.) 1980, 3 , 50-60. Maquestiau, A.; Flammang, R., Personal communication, Dec 1980. Proctor, C. J.; Brenton, A. G.; Beynon, J. H.; Kralj, B.; Marsel, J. Inf. J. Mass Spectrom. Ion Phys. 1980, 35, 393-403. Proctor, C. J.; Krai], B.; Brenton, A. G.; Beynon, J. H. Org. Mass Specfrom. 1980, 15, 819-631. Wachs, T.; Bente, P. F., III; Mckfferty, F. w. Int. J . Mass specfrom. Ion Phys. 1972, 9 , 333-341.
RECEIVED for review March 9,1981. Accepted October 8,1981. This work was supported principally by the Midwest Center for Mass Spectrometry, a National Science Foundation Regional Instrumentation Facility (Grant No. CHE78-18572), and in Part by the National Science Foundation Grants No. CHE80-08008 to M.L.G. and CHE80-11425 to R.G.C.
Effect of Inorganic Contaminants on Field Desorption Mass Spectrometry of Organic Compounds W. D. Lehmann Abtellung Medizinische Biochemie, Institut fur Physiologische Chemie, 0-2000 Hamburg 20, Federal Republic o f Germany
The influence of lnorganlc contamlnants on the sensltlvlty of organic analysis by field desorption mass spectrometry has been studied quantitatively. Several organic compounds were selected as model compounds for dlfferent processes of ion formation in fleid desorption. With the exception of glucose, the observed general tendency was a decrease in sensltlvlty with increaslng amounts of Inorganic Contaminants. The effect was found to be dependent on the salt anion and on the type of compound. As a consequence, a strategy for the chromatographlc workup of biological and environmental samples Is outlined which is adapted to the special requlrements of field desorptlon mass spectrometry.
Since the introduction of field desorption (FD) mass spectrometry (MS) (I)this novel ionization technique has had the reputation that it is difficult to obtain reproducible results. On one hand, a large variety of polar and labile model compounds were successfully investigated (2, 3) consistently showing the potential of FD MS as an analytical tool in biochemical analysis; on the other hand, in the analysis of socalled real samples extracted from environmental or biological material, experiences in the use of FD MS ranged from success without problems to complete failures (4). This variable situation most probably can be explained by nonidentical conditions mainly in two areas: firstly, the quality of the FD emitter used and, secondly, the amount and chemical nature of contaminants in the samples under investigation. A procedure for the reproducible production of high temperature activated FD carbon emitters has been recently described in detail (5). In view of the experience that FD MS is a highly specific technique for organic mixture analysis and 0003-2700/82/0354-0299$01.25/0
Universitats-Krankenhaus-Eppendorf,Martinistrasse 52,
because of the minor effects of organic contaminants on FD sensitivity, this study is concerned with the influence of inorganic contaminants on the sensitivity of organic analysis by FD MS. It can be assumed that most samples of environmental or biological origin contain a substantial and generally nondefined amount of inorganic salts originating either from the crude sample itself and/or from the work-up procedure. The pronounced sensitivity of the FD technique toward inorganic salt impurities found in this study supports the assumption that a large portion of disappointing experiences with FD MS has been caused by inorganic impurities. In the following, quantitative investigations on the extent and the variability of this salt effect are presented, and a strategy for sample purification adapted to the special requirements of FD MS is given.
EXPERIMENTAL SECTION
Mass spectrometry was performed on a VG ZAB-1F equipped with a FD ion source. All measurements were performed in the double focusing mode. The ion source potentials were +8 kV for the FD emitter and -4 kV for the counterelectrode. High temperature activated carbon emitters (6) were produced on a slightly modified VG activator according to a published procedure (5). The average length of the carbon microneedles was 30 pm. The signals were accumulated on a multichannel analyzer type Canberra series 80. For the sensitivity tests 250 ng of the compound was completely desorbed under manual heating current control and signal accumulation was performed over the entire desorption process. Comparative sensitivity tests on one compound were performed with a single emitter. The sample solution was applied to the FD emitter by use of a microliter syringe fixed in a three-dimensional micromanipulator and the application procedure was controlled under a stereomicroscope at a magnification of 63X. The variations of repeated Sensitivity tests using the same FD emitter were found to be about *20%. 0 1982 American Chemical Society
300
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982 :M+HI+
Ch2 11
COOH
'I 1 1
u w u , / & M - lI '
X 32
Flgure 1. Accumulation of the complete desorptlon of amethopterin In the pure state and contaminated with a 1000-fold molar excess of
NaCI. In each case 250 ng was desorbed from the same emitter. D-Glucose (fur biochemische Zwecke) was purchased from Merck, Darmstadt, FRG; (+)-amethopterin (Methotrexate), adenosine, and didansyl-L-tyrosine were obtained from Sigma, St. Louis, MO.
RESULTS AND DISCUSSION Sensitivity Tests on Pure and Contaminated Samples. Users of field desorption mass spectrometry are aware that inorganic salts may cause serious problems in analytical work, and, for instance, the addition of complexing agents to contaminated samples has been proposed for improvement of the desorption behavior of organic compounds (7). However, no quantitative data on the influence of this salt effect are available, and little is known about the role of the chemical nature of the contaminants. In view of the pronounced influence and of the ubiquitous presence of inorganic salts in samples of biological origin, we have attempted quantitative studies on this effect. Therefore FD sensitivity was tested on samples with a defined content of both the organic compound and the inorganic constituents at varying molar ratios of organic compound to inorganic salt. First, amethopterin was selected as a strongly polar compound showing an abundant [M + HI+ ion in FD MS and only a few fragment ions of minor intensity in addition. Figure 1 shows the result obtained in the analysis of amethopterin when a dilute solution in pure methanol was contaminated by a stepwise addition of definite amounts of sodium chloride. As shown in Figure 1, a molar excess of 3 orders of magnitude of sodium chloride reduces the efficiency of the [M + H]+ formation to about 3% of the value observed for the pure compound. In general, the reduction of the absolute sensitivity observed with the presence of inorganic salts is accompanied by two additional effects: the range of heating currents, in which a desorption is observed, is shifted by several milliamperes to higher values and the relative intensities of fragment ions are strongly enhanced. Quantitative investigations in this study have been confined to the production of molecular ion species, as the detection of these types of ions represents the most important aim in analytical applications of FD MS. With respect to samples purified by a chromatographic process, the presence of a buffer salt, such as a phosphate salt, is a more realistic example than a contamination with sodium
Figure 2. Accumulation of the complete desorption of 250 ng of amethopterin: (a)pure compound, (b) contaminated wlth an equimolar amount of NaH,PO,, (c) contaminated with a IO-fold molar excess of NaH,PO,.
chloride. In the study of the addition of phoshates we generally observed that these exhibit a more negative effect on FD ion production than salts with singly charged anions, such as chloride, bromide, and iodide. Figure 2 gives a comparison of the absolute intensities obtained with a sample of amethopterin contaminated with different amounts of sodium dihydrogen phosphate. Compared to the results given in Figure 1it is evident that the presence of sodium dihydrogen phosphate has a more serious effect than has the presence of sodium chloride. A molar ratio of 1:l (organic compoundsalt) results in a decrease of sensitivity, and a 10-fold molar excess of the salt practically prevents success. In contrast to the desorption behavior in Figure 2a,b, characterized by smooth and long-standing desorption, the desorption connected with Figure 2c showed strong fluctuations, the majority of the sample desorbing within a few seconds. Under the conditions of explosive desorption behavior the use of a photographic detection system probably would have provided a clear and intense mass spectrum. A broad variety of compounds extracted from environmental or biological samples has been analyzed by FD MS and photographic registration (2, 3). A tendency similar to that of amethopterin was observed for adenosine. Again, the absolute sensitivity was reduced by the addition of salt and contamination with a phosphate salt had a more negative effect than contamination with the same number of moles of sodium chloride. The investigations with adenosine also supported another result which was already evident from the spectra in Figures l and 2: the [M + H]+/[M + Na]+ ratio does not reflect the sodium content of the sample even if the same emitter is used throughout. Thus, the salt content must be determined separately. For relatively volatile compounds that predominantly form [MI+' ions in FD MS, a pronounced suppression of ion production after the addition of salt was also observed. This is illustrated in Figure 3 for didansyl-L-tyrosine analyzed as the pure compound and as mixtures with NaCl (molar ratios of 1:1, l : l O , and 150). The addition of an equimolar amount of sodium chloride does not alter the absolute sensitivity observed for di-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
301
[M*H]*
[MI’’ m / z 647
’
mlz 285
\ I
ii
‘ I
&.&A l h - L . . & + A h , ~ b ~ h’ * i ,
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x 8
I
CI
dd-
x 8
-L+-
Figure 8. Accumulation of the complete desorption of 250 ng of didansyk-tyrosine: (a) pure compound, (b) contaminated with a molar excess of 1:lO of NaCI, (c) Contaminated with a molar excess of 150 of NaCI.
I
1
OH
IM*Nal+ m/z 203
1’I 1
IMtH: m/z 181 \
m/z 18: \
1
A
NoCl 1 : l
ibm pure
Flgure 4. Accumulation of the complete desorption of 250 ng of pglucose: (upper trace) contaminated with NaCl at a molar ratio of 1:l; (lower trace) pure compound.
dansyl-L-tyrosine significantly. A 10-fold molar excess, however, reduces the ionization efficiency by about l order of magnitude (Figure 3b) and a molar ratio of 1:50 as shown in Figure 3c allows the detection of only a few percent of the original ion current, with elevated background and strongly reduced resolution. The three compounds presented so far have been selected as model compounds for different types of behavior under FD conditions, namely, for the predominant formation of [M H]+ ions without a strong tendency for cationization by alkali metals (amethopterin), for the competing formation of [MI+’ and [M H]+ ions (adenosine), and for the prevailing formation of [MI+. ions (didansyl-L-tyrosine). For all three compounds the highest sensitivities were achieved when the pure compounds were analyzed. To a first approximation, the addition of salt up to a ratio of about 1:l was tolerated without adverse effects; but further increase led to a decrease in sensitivity which depended on the salt anion and on the type of compound. Different behavior was observed for glucose. Glucose is a model compound for substances which only form cationized molecules with an affinity to alkali cations rather than to protons. Here the pure compound gave the [M + HI+ ion as base peak accompanied by a very small [M + Na]+ signal. A 1:lmixture with NaCl gave a spectrum in which the relative intensities were reversed; the absolute intensity of the [M + Na]+ ion compared to the [M + H]+ ion in the investigation of the pure compound was increased by a factor of about 30, as shown in Figure 4. For polyhydroxy compounds such as sugars the presence of an equimolar amount of salt can enhance FD sensitivity. For increasing amounts of NaCl a loss in sensitivity was also observed in this case. Reproducible results as obtained in the
+
+
Figure 5. Protonated molecule of 4-hydroxyantipyrine sulfate recorded by FD MS and electrical detection. A total of 60 repetitive sweeps were accumulated at 18-22 mA emitter heating current.
investigation of the other model compounds could not be produced for glucose in this study. Another exception to the simplified summary given above was antipyrine sulfate, an important conjugate of antipyrine isolated from rat urine (8,9).After extraction from rat urine this compound was purified by column chromatography and then was subjected to a fiial purification by HPLC with a pure water/methanol system. Investigation of the HPLC eluate fraction containing the sulfate conjugate as alkali salt yielded no ionic species containing the intact sulfate ester. Then, the sample was desalted by ion exchange chromatography on a strongly acidic cation exchange column and the free acid was applied to the FD emitter. Now the FD mass spectrum given in Figure 5 was obtained showing the [M + HI+ ion of this highly polar and labile conjugate as the most abundant ion signal. In this case a molar ratio of about 1:l between organic compound and alkali cations, as in sodium 4-hydroxyantipyrinesulfate, already was sufficient to suppress the desired smooth desorption of [M HI+ ions. Field desorption mass spectra of sodium salts of sulfate and phosphate esters showing intact molecules cationized by sodium have been reported (10, 11);however, these spectra were recorded photographically. For the reduction of the FD sensitivity with increasing concentration of alkali cations we see the following main reasons: An excess of inorganic salts covers the organic constituents in a salt matrix, in which the organic molecules are partly pyrolyzed when the emitter heating current is increased. The surface mobility of the sample molecules is reduced, leading to a less efficient sample supply to the ionizing centers and possibly to less efficient ionization reactions on the emitter surface (12). The salt layer reduces the average field strength on the emitter surface by covering microneedle tips. Through solvent occlusion the sample solution does not dry completely on the emitter surface. Thus, explosive desorption is observed being highly unfavorable for electrical detection of FD mass spectra. Further work will be required to clarify the relative importance of these adverse effects on ion production in FD MS. Estimation of Salt Contamination by FD MS. A large molar excess of inorganic salts in an organic sample can be recognized qualitatively when the emitter is heated quickly from about 35 to 100 mA to purify the emitter wire. The metal cations desorb from the FD emitter, causing a strong leakage current between emitter and counterelectrode. FD can also quantify the salts present in the sample, as the technique is characterized by an outstanding sensitivity for a variety of metal cations (e.g., ref 13 and 14). It can be assumed that sodium and potassium represent the vast majority of metal cations in a purified sample of organic material, a situation
+
302
ANALYTICAL CHEMISTRY, VOL. 54, NO. 2, FEBRUARY 1982
Table I. Estimation of Molar Ratios between Organic Compounds and Salts in Liquid Chromatographic Separations inorganic buffer concn eluate
lo-' lo-' lo-'
mol/L mol/L mol/L mol/L
salt/mL of molar eluate org compd/mL of ratio org (mol wt eluate (mol wt compd/ 100) 400) salt 1 0 pmol 10 pmol 1 pmol 1 pmol 10 nmol' 1 0 nmol'
1pg ( 2 . 5 nmol) 10 ng ( 2 5 pmol) 1 ug (2.5 nmol) 10 ng ( 2 5 pmol) 1 pg ( 2 . 5 nmol) 1 0 ng (25 pmol)
2.5:104 2.5:106 2.5:103 2.5:105 2.5:lO 2.5:103
Estimated on the basis of a salt contamination of 1 PPm. a
which reduces the problem to a quantification of these two metals. The method of choice for quantification by FD MS, as for all other mass spectrometric techniques, is internal standardization with stable isotope labeled analogues. This principle excludes the exact quantification of monoisotopic elements such as sodium. However, with reduced accuracy sodium can be quantified relative to ita homologue potassium. Repeated complete desorptions of standard mixtures of NaCl and KCl (molar ratios of 1:1,101, and 1OO:l)showed that an analysis by relative peak heights results in an overestimation of potassium relative to sodium with a factor between 2 and 5. Taking this factor into account, the correct order of magnitude for the amount of sodiqn can be estimated relative to the signal height of potassium, which can be quantified accurately by the addition of 41K as internal standard. The Combined Use of HPLC and F D MS. In order to use the results given above to derive consequences for the combined use of HPLC and field desorption MS, we first consider the molar ratios of organic compounds to salt in a HPLC eluate. Table I summarizes the data calculated for 1 mL of a HPLC eluate containing a nonvolatile buffer system a t typical concentrations or a volatile (or no) buffer. The direct analysis of a HPLC eluate containing a nonvolatile buffer system clearly is not advisable; for microgram amounts of organic compounds a molar excess of salt of 105-104is expected. On the other hand, the use of a volatile buffer system will result in a roughly equimolar amount of inorganic salt in the eluate fraction. For many classes of compounds this degree of purity will be sufficient for a near-optimal analysis by FD MS. Accordingly, in combined applications of HPLC and FD MS the chromatographic separations have been based either on a system of pure solvents (15-17) or on a volatile buffer system (18, 19) which could be removed before the mass spectrometric analysis. However, the amounts of salts given in Table I represent minimum values as here the HPLC solvent system was regarded as the only source of alkali salt contamination. In practice, additional amounts of salta may be introduced during sample handling or by incomplete removal of the salts originally present in the crude sample. In these cases an additional purification step will be necessary. A common procedure to effect a reduction in inorganic salts is a solvent extraction using an organic solvent such as methylene chloride, chloroform, ethyl acetate, or a mixture of these. For instance, if the dry residue of the contaminated sample of didansyl-L-tyrosine shown in Figure 3c is redissolved in chloroform, the FD spectrum of Figure 3a is obtained. Thus, the optimal sensitivity as obtained for the pure compound can be nearly reinstated by this simple solvent extraction. If this procedure does not give a sample of sufficient purity, chromatography with pure solvents, an ion exchange
WITH NONVOLATILE BUFFER
j
WITHOUT B U F F E R
I*
- 1
ION EXCHANGE CHROMATOGRAPHY
+
t FIELD
DESORPTION
MASS
i
SPECTROMETRY
Flgure 8. Recommended work-up procedures for the comblned use of liquid chromatography and field desorption mass spectrometry. 1285s
;*EE"5 248 F i E S E T = 2ZBZ ~
:#E-L
~
UNIT# 1 T A G NC.
1 MILLISECOND
e ca'*
vcs- ? ? ? 2 7 2
CRT-(Ol-ZB)
:123 I
3 J-N E--
-
81
183032 2 0 4 7 CH#
Flgure 7. Quantification of r-tyrosine from a sample of human plasma (79). About 20 ng of dldansyl-r-tyrosinewas applied to the emitter
and 248 sweeps were accumulated at current.
24-30 mA
emitter heating
step, or a combination of both has to be performed. Figure 6 schematically summarizes recommended sequences of sample purifications which are adapted to the requirements of FD MS. The procedures given in Figure 6 lead to a stepwise reduction of the contamination by inorganic salts. As samples of biological or environmental origin even after purification often contain more salt than would be desired for an optimal analysis by FD, this concept of a stepwise purification given in Figure 6 clearly is to be preferred over the concept of adding inorganic or organic compounds to overcome the negative influences of contaminants. Absolute Sensitivity of F D MS. Interestingly, the optimal sensitivities observed in numerous investigations for the five compounds amethopterin, adenosine, didansyl-L-tyrosine, cholesterol, and D-glucose fall within the relatively small range of (0.5-2) X C/fig. Thus it appears probable that the sensitivity of FD MS, in a first approximation, does not depend on the prevailing process of ion formation, which can result in the formation of [MI+.,[M + HI+, or [M + Cat]+ ion species where [Cat]+ represents a metal cation. This sensitivity enables the recording of a clear mass spectrum containing quantitative information from nanogram amounts of samples. As a demonstration, Figure 7 shows a FD mass spectrum recorded from a sample of 20 ng of didansyl-L-tyrosine, from which the plasma free L-tyrosine level in a sample of human blood can be calculated (19). These estimations show that FD MS operated under optimal conditions is a valuable tool in organic trace analysis. However, the reduction of its sensitivity by a factor of 10 or more caused by nonoptimal conditions greatly reduces its value for analysis, thus underlining the importance of the effective removal of inorganic salts. ACKNOWLEDGMENT I am indebted to H. M. Schiebel and N. Theobald for numerous valuable discussions. The sample of 4-hydroxyantipyrine sulfate was kindly provided by J. Bottcher, Institut
Anal. Chem. 1982, 5 4 , 303-307
fur Pharmakologie und Toxikologie, Technische Universitat Braunschweig.
LITERATURE CITED (1) Beckey, H. D. Int. J. Mass Spectrom. Ion Phys. 1989, 2 , 500. (2) Schulten. H A . I n "Methods of Biochemical Analysis"; Gllck, D., Ed.; 1977; VOI. 24, pp 313-448. (3) Schulten, H A . Int. J. Mass Spectrom. Ion Phys. 1979, 32, 97. (4) Fenseiau, C. Biomed. Mass Spectrom. 1978, 3 , 149. (5) Lehmann, W. D.; Fischer, R. Anal. Chem. 1981, 53, 743. (6) Schulten, H A ; Beckey, H. D. Org. Mass Spectrom. 1972, 6 , 885. (7) Wood, 0. W.; Lau, P.-Y.; Mak, N. Biomed. Mass Spectrom. 1974, 1, 425. (8) Bottcher, J.; Baszmann, H. M.; Schuppel, R. Drug Metab. Dlspos ., In press. (9) Lehmann, W. D.; Schlebel, H. M.; Bottcher, J.; Baszmann, H. M.; Schuppel, R. Biomed. Mass Spectrom., In preparation.
303
(10) Schulten, H A . ; Beckey, H. D.; Bessell, E. M.; Foster, A. B.; Jarman, M.; Westwood, J. H. J. Chem. SOC.,Chem. Commun. 1973, 13, 416. (11) Schulten, HA.; Lehmann, W. D. Anal. Chim. Acta 1978, 8 7 , 103. (12) . . Holland, J. F.; Soltman, B.; Sweeley, C. C. Blomed. Mass Spectrom. 1978, 3 , 340. (13) Lehmann, W. D.; Schulten, H.-R. Anal. Chem. 1977. 49, 1744. (14) Lehmann, W. D.; Bahr, U.; Schulten, H.-R. Homed. Mass Spectrom. 1978, 5, 536. (15) Schulten, H A ; Beckey, H. D. J. Chromatogr. 1973, 83, 315. (16) Schulten, H A ; Kbrnmler, D. Anal. Chlm. Acta 1980, 713, 253. (17) Stober, I.; Schulten, H.4. Sci. TotalEnviron. 1980, 76, 249. (18) Deslderlo, D. M.; Cunnlngham, M. D. J. Llq. Chromatogr. 1981, 4 , 721. (19) Lehmann, W. D.; Theobald, N.; Helnrich, H. C. Biomed. Mass Spectrom. 1981, 8, 0000.
RECEIVED for review July 27,1981.Accepted October 6,1981.
Potentiometric Determination of Nicotinamide Adenine Dinucleotide Phosphate and Glutathione Reductase Saad S. M. Hassan' and G. A. Rechnltr" Department of Chemistry, University of Delaware, Newark, Delaware 1971 1
A carbon dloxide gas senslng membrane electrode Is employed for the potentlometrlc determlnatlon of NADP' and glutathione reductase enzyme using a rate approach. The resulting methods comblne convenlence, sensltivlty, and high selectlvlty with good analytical preclslon and accuracy. It Is also shown, through the enzymatic cycllng scheme employed for the glutathione reductase determination, that the potentlometric method Is suited for the In sltu monkorlng of NADP' levels without Interference by NADPH.
Nicotinamide adenine dinucleotides are important cofactors for a large number of oxidoreductase enzymes functioning in metabolic processes (1). The levels of these nucleotides are usually monitored by a number of spectroscopic and electrochemical methods. The change in the absorbance (2,3) or fluorescence (3-5)at 340 nm due to nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) and the color developed at 510 nm by a reaction with p-dimethylaminobenzaldehyde (6) have been used for their quantitation. In the presence of methylene blue, NADH reduces oxygen to hydrogen peroxide which is then determined by a chemiluminescence method through a reaction with bis(2,4,6-trichlorophenyl)oxalatein the presence of perylene (7). Amperometric measurements by using a polarizing potential (8) or by coupling to various redox reagents (9-11) or enzymes (12) have been reported. Voltammetric determination at a carbon paste or glassy carbon electrodes has also been utilized (13,14). The lower limits of detection offered by the spectroscopic and electrochemical methods are 10-6-10-8M and 104-104 M, respectively. However, most of these methods either are inapplicable to turbid or colored solutions or suffer from the inability to differentiate between NADH and NADPH. A number of cyclic procedures has also been developed for the measurement of the pyridine nucleotides down to 'On leave from Ain Shams University, Cairo, Egypt. 0003-2700/82/0354-0303$01.25/0
M levels. These are based on the use of two enzymatic reactions to bring about consecutive oxidation and reduction of the nucleotides in a cyclic fashion. After sufficient cycling, one of the products is measured by spectrophotometric (15-17) or fluorometric (18)techniques or by using a third enzyme to give a measurable species (19,ZO). Among the most sensitive methods are those involving the use of lactate and glutamate dehydrogenases to convert the 14C-labeledlactate in the presence of NADH or NAD+ into I4C-labeledpyruvate followed by decarboxylation with pyruvate decarboxylase (19). Conversion of 14C-labeleda-ketoglutarate by lactate-glutamate dehydrogenase as a function of NADPH or NADP+ into 14Clabeled glutamic acid and subsequent decarboxylation by the action of glutamic decarboxylase has also been reported (20). The l4COZreleased from these reactions is counted as a measure of the nucleotide concentrations (19,ZO). Although a high sensitivity is gained by this approach, these methods are far from being selective. Both the oxidized and reduced forms of the nucleotides are recycled in all the reactions described (15-20)rendering the measurement of one species in the presence of the other impossible without prior chemical treatment (18)or separation by liquid chromatography (21). These cyclic methods are also not suitable for the measurement of the rate of formation or consumption of one form of the nucleotides in the presence of the other. Moreover, some of these procedures require several manipulation steps, the use of radioactive reagent, and sophisticated instruments. On the other hand, the literature reveals that malate dehydrogenase decarboxylating enzyme selectively catalyzes the decomposition of L-malate substrate in the presence of NADP+ coenzyme with the formation of pyruvate, GOz, and NADPH. The kinetics of this reaction has been investigated (22,23) and the rate of COz liberation or NADPH formation has been successfully monitored as a function of the enzyme activity (24-26). The present investigation was undertaken to develop a simple and selective method, based on this reaction, for the potentiometric determination of NADP+ under conditions where both NADP+ and NADPH coexist and, further, to follow the rate of NADP+ formation in situ as a function of 0 1982 American Chemical Society
