Differential Pulse Polarographic Determination of Some Carcinogenic Nitrosamines Kiyoshi Hasebe and Janet Osteryoung Departments of Civil Engineering and Microbiology, Colorado State University, Fort Collins, Colo. 80523
The pulse polarographic behavior of N-nltrosopyrrolidine and two of its derivatives, N-nitrosoproline and Knitroso4-hydroxyproline, has been investigated in buffered solutions over a range of pH. The effects of temperature, mercury pressure, modulation amplitude, surfactant concentration, and solvent and supporting electrolyte have been studled. The reduction process is complicated and pH-dependent. In acid solutions, Knitrosopyrrolidlne and its derlvatlves give one irreversible reduction wave, while for pH greater than 6, only N-nitrosopyrrolidine Itself is reduced. The reduction waves of Knitrosoproline and Knitroso-4hydroxyproline at pH below 5 are dlffusion-controlled in dc and normal pulse polarographic modes. In aqueous medium, N-nitrosopyrrolidine, Knitrosoprollne, and Knltroso-4-hydroxyproline have been detected quantitatively at the M level by differential pulse polarography with adequate precision and accuracy. The Importance of examining electrochemical behavior to aid in selecting Conditions for analysis is discussed.
Secondary amines react with nitrite under acid conditions to form N-nitrosamines, many of which are carcinogenic, mutagenic, or teratogenic (1-4). Secondary amines are common constituents of foodstuffs and can react with naturally occurring or added nitrite under food processing or storage conditions or in the stomach to form N-nitrosamines. The most important potential human exposure to these compounds is in cured meat products. Studies of the prevalance, toxicity, and significance to public health of these compounds requires analyses a t the 5-10 pg/kg level which corresponds roughly to solutions a t the lo-' M level. Many analytical methods such as spectrophotometry (5), gas-liquid chromatography (6, 7 ) , and gas chromatography-mass spectrometry (8) have already been applied to the determination of N-nitrosamines. Of these techniques, only the GC-MS approach now provides both qualitative and quantitative information sufficiently reliable for routine analytical operation, but the GC-MS analysis is time consuming and expensive and can be applied only to volatile compounds. In this paper, we describe the pulse polarographic behavior of some nitrosamines and report suitable conditions for analysis. The nitrosamines studied are N-nitrosopyrrolidine (NOPyr) and two of its derivatives, N-nitrosoproline (NOPro) and N-nitroso-4-hydroxyproline (NOHOPro). NOPro is relatively nonvolatile and unstable with respect to temperature. It melts with decomposition a t 100 "C and on mild heating of a slightly alkaline aqueous solution decarboxylates to form NOPyr. NOPro is not carcinogenic, but is found in most fatty cured meats (e.g., bacon), and its decarboxylation product, NOPyr, is one of the most potent carcinogens so far identified. The decarboxylation reaction apparently takes place to some extent under meat processing conditions (9, 10). The 4-hydroxy derivative is also of interest because the precursor amino acid occurs free in foods. 2412
There is little information about polarographic analysis for N-nitrosamines in the literature (11, 12). In trace analysis, dc polarography has insufficient sensitivity and ac polarography is usually limited by the irreversibility of the electrode process. Pulse polarography is a well-established electrochemical technique which minimizes ac capacitative current and, especially in the differential mode, is proving to be a versatile technique for trace determination of metals and nonmetals (13, 14). Pulse polarography has the advantage of providing sensitive, rapid analysis with simple, inexpensive equipment, and in principle has less demanding sample cleanup requirements than the GC-MS technique. The differential pulse (DP) mode peak current provides lower detection limits than does the normal pulse (NP) mode limiting current, but the former is more sensitive to solution conditions, for its magnitude depends on the kinetics of the electrochemical process. The polarographic reduction of some N-nitrosamines has already been studied, mainly from the point of view of establishinF reaction mechanisms (15-21). In particular, Zahradnik et al. have studied both the spectroscopic and dc polarographic properties of the compounds of this work (21). In general, a t mercury electrodes N-nitrosamines exhibit an irreversible four-electron reduction in acid solution to the corresponding unsymmetric hydrazine and an irreversible two-electron reduction in basic solutipn to nitrous oxide and the precursor amine (17).Zahradnik et al. have described the reduction process in acid solution as the sequence of steps: reactant adsorption, protonation of the adsorbed species, electrochemical reduction, and follow-up chemical and electrochemical steps (21). The reduction in basic solution may also involve adsorption, but is p H independent. For the purposes of analytical method development, therefore, we have determined the following: the conditions under which the N P limiting current is diffusion controlled and therefore insensitive to small changes in conditions, the effects of p H on N P and D P currents, the effects of some solvents, supporting electrolytes, and surface active substances on reactant adsorption and hence on the reduction kinetics and the D P or N P currents, and the effects of instrumental parameters such as pulse height in the D P mode. A t the same time, we have examined the effects of solution parameters on dc capacitative background currents because, especially in the D P mode, these currents are large and play an important role in determining the detection limit (22, 23). Finally, we have determined sensitivities and detection limits for these compounds under representative sets of good conditions.
EXPERIMENTAL Polarographic data were obtained with a Model 174 Polarographic Analyzer (Princeton Applied Research Corporation, Princeton, N.J.) and a Model 174/70 Drop Timer. The polarograms were recorded on an Omnigraphic Model 2000 X-Y recorder (Houston Instrument Company, Austin, Texas). The dropping mercury electrode used had the following characteristics: mercury flow rate m = 1.92 mg sec-' in deionized water at open circuit and natural drop time t d = 4.60 sec in 0.1 M lithium chloride containing 50 v/v% Britton-Robinson buffer at -0.60 V vs. SCE, and at a
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
height of the mercury reservoir hUnCOrI = 43.0 cm. The cell was a covered 100-ml Berzelius beaker with carbon rod counter electrode. A presaturated purified nitrogen stream was used to deoxygenate the solutions and to keep them oxygen-free. The effluent gas was passed through a trap containing concentrated HC1 and glacial acetic acid (5:l) in which the nitrosamines are degraded ( 2 4 ) .Measurements of pH were made with a Fisher pH/ion meter Model 230 or a Corning Model 110 expanded scale digital pH meter. Except for temperature dependence studies, all measurements were carried out at room temperature kept constant at 20 f 1 O C . The chemicals used were reagent grade and were dissolved in deionized water. N-Nitrosopyrrolidine (NOPyr) was obtained from Aldrich Chemical Company, Inc. N-Nitrosoproline (NOPro) and N-nitroso-4-hydroxyproline (NOHOPro) were prepared from L-proline and 4-hydroxy-~-proline(both chemicals 99+% Gold Label, Aldrich Chemical Company, Inc.), respectively. N-Nitrosoproline was synthesized as follows. To 11.5 mg of L(-)-proline in about 80 ml of 0.1 M hydrochloric acid, 0.69 g of sodium nitrite was added slowly with cooling in a cold water bath. After reacting 1 day, the mixture was accurately diluted to 100 ml with 0.1 M hydrochloric acid. N-Nitroso-4-hydroxyproline was synthesized similarly. NOPro crystals were prepared according to a modification of the method of Lijinsky et al. (251, Le., 6 g of Lproline was dissolved in HC1 (4ml) and water (20 ml) with cooling in a water-ice bath, and 5 g of NaN02 was added slowly. After 1 hr, the water was evaporated from the reaction mixture using a rotary evaporator, and NOPro was extracted with pure acetone. T o this extract, 5 g of silica gel (60-200 mesh) and 2 g of activated charcoal (60-200 mesh) were added, and then filtered off. Acetone was removed by evaporation in a cold bath with a stream of Nz. The first crop of crystals was recrystallized from chloroform. The white crystalline product was identified and purity established by NMR spectra, ir spectra, uv spectra, and melting point measurements. The molar absorptivity at the uv maximum, 238 nm, was M aqueous HC1 and Beer's law was found to be 6700 f 100 in obeyed over the range 0.9 X 10-4-1.5 X M . The molar absorptivity may be compared with the vaiues reported by Lijinsky et al. ( 2 5 ) ,6500, and by Zahradnik et al. (21), 6000. N-Nitrosamine stock solutions were prepared in 0.1 M HCl. These solutions were found to be stable as determined by polarography for periods exceeding 4 months. The pH of the polarographic solutions was adjusted with Britton-Robinson buffer rn the pH range from 1.8 to 11.9. Below about pH 1, the measurements were made in HzS04 or HCl. Stock solutions of the synthesized nitrosamines prepared directly from the precursor amine contain electroactive impurities which do not interfere with the electrochemical nitrosamine reductions. Based on spectrophotometric measurements, the nitrite concentration in these solutions is about 100 times the nitrosamine concentration. In acid solution, nitrous acid disproportionates to nitric oxide and nitrate and the former is removed by the purging gas.
RESULT AND DISCUSSION Effects of pH. T h e effects of p H on the reductions are shown in Figures 1, 2, and 3 and in Table I. T h e data are in general accord with results on other N-nitrosamines and support the mechanism of reduction of the protonated form in acid solution. Free NOPyr is directly reduced in basic solution. However, in the same p H range, NOPro and NOHOPro are present only as anions and therefore are so irreversibly reduced that tke waves are not well separated from background. Zahradnik e t al. report the p H independent reduction of NOPro and NOHOPro in borate buffers a t approximately -1.8 V vs. SCE (21). Since the NOPyr reduction is about 300 mV more anodic, this makes it possible t o analyze mixtures of, for instance, NOPyr and NOPro by determining the total concentration in acid solution and the NOPyr concentration alone in basic solution. The slopes of the potential vs. p H curves are consistent with involvement of one proton per electron in the rate determining step. Complications of adsorption account for the large values of the slopes for this process, Normal pulse and d c currents are constant over a wider p H range for NOPyr than for NOPro or NOHOPro. This is possibly due to the formation of the anions of the latter
. P A.
E30
IP
InP
15 id, 7 6
1.10
cn 111
10,5
6.
W
I
c m
4
3
W
I
3 5 0.70
2
1 050
0
2
4
6
8
10
0
-50 mV
PA IP,
in,
2my
0 dc 0 dPP
0 "PP
1.50
>
idc
15, 3
10,2
n
P
W
I
b
>?
W
I
1
5, 1
0.50'
0
I
I
2
4
6
8
loo
Figure 2. Effect of pH on the wave heights and half-wave or peak potentials for Nnitroso-4-hydroxyproline 1.00 X
MNOHOPro; other
conditions same as Figure 1
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2413
L60 -
1.40-
1
-20, 20, 10
P
> 1.20npp
% -
-----
22
W I
-10, 5
1.00-
0.80 0
1
3
2
6
5
4
7
8
B
11
10
1 0 0 12
PH
Figure 3. Effect of pH on the wave heights and half-wave or peak potentials for Knitrosopyrrolidine M NOPyr. Other conditions same as Figure 1
1.08 X lo-'
compounds. The pK, for the carboxylic acid group of NOPro is 3.0 and that for NOHOPro is 2.9 (25). Note also in Figure 2, the change in slope of the Ell2 vs. pH curve a t pH 2.8. The dc current for reduction of NOHOPro is substantially less a t pH 0 in HC1 than in the range pH 1.0-2.4. This is associated with a large background current and, though reproducible, seems to be an artifact associated with the supporting electrolyte. The same effect is not seen in H2SOl Table I. Polarographic Characteristics of N-nitrosamines Compound
NOPyr
NOPro
NOHOPro
0.93
1.16 0.97
0.97 0.72
Logarithmic analysis (dc)g
(pH 1.9)b (pH 2.35) (PH 8.5) (pH 1 . 8 ) a
1.00
0.54
...
Hg-head dependence-hP
DC (pH 1.9); NP (pH 1.9) DC (pH 9.1)c NP (pH 9.1)d DC (pH 1.75)a,c NP (pH 1.75)a#d Relative temp. coeff. at 2 0
DC NP DP DCa
U"/1 of DPe,
... ...
,
P
0.47 0.73 0.51 0.63
... ...
"C (pH
0.50 0.66
... ...
0.47 0.68 1.49-2.00)
1.52 1.57 1.06
...
E% vs. pH
DC NP DPf
...
1.16
79 84 84
in
1.64
... ...
0.51 0.67
... ... ...
...
water, %'OK-' 1.39 1.49 1.60
1.93 mV/pH
...
137 160 160
130 130 110
110 105 145 mV a 20 v/v % EtOH. naa = 0.97 for Tl+. ' p = 0.47 for Tl+ p = 0.66 for T1+. e AE = -50 mV; pH 1.9. f E p . g n a a = (2.303RT/F)[Alog(id - i.)/i]/AE.
2414
solution. On the other hand, the decrease in current a t low pH for NOPyr in the D P mode is undoubtedly real and due to a decrease in the reduction rate under these conditions. Experiments at lower pH values continue this trend. It is most important analytically that the D P current is very sensitive to changes in rate and mechanism, and therefore may change in unusual and unpredictable ways even when the N P or dc currents are well behaved. Reversibility. The N P and dc polarographic waves were analyzed logarithmically to obtain values of naa.Some representative results for the dc waves are shown in Table I. In acid solution, the naa values might correspond to n, = 2, a 0.5. The low value for NOHOPro a t pH 2.35 is clearly reflected by the change in the D P peak current shown in Figure 2. In basic solution for NOPyr, possibly n, = 1 and a N 0.5. These values may be compared with the D P peak widths (Table I) which are larger the more irreversible the reaction. The peak width for a reversible one-electron reduction at AI3 = -50 mV is 100 mV. The determination that the reduction is irreversible shows that the D P current will be less than the reversible current and that the current-concentration relation will depend on rate parameters. This makes it imperative to identify variables, especially those of solution composition, which affect the rate of the reduction process. Diffusion Control. Dependence of limiting current on mercury head has been studied in the dc, NP, and D P polarographic modes. For diffusion-controlled waves, the height of the dc or N P wave is approximately proportional to h:L:r when the natural drop time is used, and to hz!?,with a mechanically controlled drop time. Table I shows the value of p obtained by plotting values of log i vs. log h to obtain the power of h. The results generally indicate that the limiting currents are diffusion-controlled. Data for NOPyr are shown in Table 11. The standard deviation of these values lies in the range of values reported (0.023-0.193) in the literature (26). The reductions are also diffusion controlled according to criteria of the t116 dependence of the current on individual drops and the agreement of the ratio of limiting currenJs in dc and N P modes with the theoretical value. Zahradnik et al. have also reported diffusion control in the dc mode (21). Therefore, the diffusion coefficients of these N-nitrosamines were estimated by using the dc polarographic data and the Ilkovic equation, taking into account the variation
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
Table 11. Effect of Height of the Mercury Reservoir on the dc and Normal Pulse Polarographic Wavesa h, cm
28.0 33.0 43.0 53.0 63.0 73.0 Mean Std dev ( s )
idc, P A
inp, pA
0.81 0.89
2.19 2.50 2.88 3.34
1.00 1.12
1.24 1.34
idc/h'12 COrr (FA cm-*")
0.158, 0.159, 0.156, 0.156, 0.158, 0.159, 0.158, 0.001,
Table 111. Diffusion Coefficients
De polarizer PH D Q (em2 sec-' x i o 5 ) T1' 1.98 8.00 0.12 inp/h213corr NOPro 1.96 0.752 t 0.035 (PA ~ r n - ~ ' ~ ) NOHOPro 1.96 0.669 f 0.031 NOPyr 9.09 1.19 k 0.17 0.249, NOPro 1.9oc 0.349 k 0.009 0.253, NOProd 2.10 0.856 F 0.064 0.242, The true value of these coefficients may be expected to 0.242, lie within the range above mentioned with a 99% probability. b Supporting electrolyte: 0.1 M LiCl + B-R buffer. C 20 v/v % EtOH. d Based on solution prepared with syn0.246, thesized NOPro crystals. 0.004,
*
Q
a 1.083 x lo-' M N-nitrosopyrrolidine, 0.1 M LiCl containing 50 v / v % Britton-Robinson buffer (pH 9.09). Ionic strength, ca. 0.11.
of drop time. The results are shown in Table 111. T h e diffusion coefficient of T1+ ion was measured under the same conditions to provide a check on the results, and gives excellent agreement with the published value of 2.00 X 10-5 cm2 sec-l (27). The value of D for NOPyr is somewhat larger than that of the others, but there is no reason to suspect experimental error in this value. Two values are given for D for NOPro, one based on a standard solution of proline converted to NOPro and one based on a standard solution of NOPro prepared from synthesized NOPro. The concentration of the second solution is more accurately known. However, both values are given because all other data for NOPro are based on the first solution. All absolute currents reported for NOPro should be multiplied by the ratio of the square roots of these D values, 1.067. T e m p e r a t u r e Dependence. The temperature dependence of the polarographic reduction of the N-nitrosamines has been studied. As is well-known, half-wave potentials of reversible processes are nearly independent of the temperature. While the N-nitrosamines studied in this work are irreversible systems, Ell2 or E , of NOPro observed in mixtures containing 44 v/v % ethanol (apparent p H 2.45) and E1/2 or E , of the same compound in mixtures containing 20 v/v YO ethanol (apparent pH 1.90) were nearly independent of the temperature within experimental error. Also or E , of NOPyr in aqueous solutions was also temperature independent. This result supports the view that the reduction rate depends on adsorption of reactant, for decrease in the extent of adsorption with increasing temperature tends to cancel out the general rate enhancing effect of increasing temperature. Some of the relative temperature coefficients of the current for N-nitrosamine reduction in the temperature range from 1 to 44 "C are shown in Table I. These results correspond to activation energies of diffusion and therefore are typical of diffusion-controlled reactions. Zahradnik et al. report temperature coefficients of about 2.5%. Effect of O r g a n i c Solvents. If miscible organic solvents are present in the polarographic solution and there are no resulting specific chemical effects, the limiting current should decrease due to changes in the drop time and the diffusion coefficient with changing ionic strength and the viscosity of the medium. Figure 4 shows the effect of ethanol on the limiting current and half-wave or peak potential for the reduction of NOPro. A similar phenomenon has also been observed in the cases of the other N-nitrosamines in alcohol-water mixtures. For example, the wave height or peak current of NOPyr in polarographic solutions containing 20 v/v % ethanol is about 45% of that in the aqueous solution only. It is important to note that the presence of the organic solvent in the polarographic solution decreases the
sensitivity. However, the reduction of NOPro and NOHOPro is also diffusion-controlled in ethanol- or methanolwater mixtures as it is in water by criteria of the relative temperature coefficient and the mercury head dependence of the limiting current, as shown in Table I and Figure 4. The N P and dc currents in ethanol-water mixtures are those to be expected from the change in viscosity of the solvent. The currents relative to current in water at 20 "C are all about 0.04 larger than those predicted from viscosity data for 0.1 F LiBr in ethanol-water mixtures a t 25 "C (28). On the other hand, the currents in methanol-water mixtures are much larger than those predicted from similar viscosity data. Over the range 0-30% methanol, the decrease in current is only about 5% while the square root of the viscosity for 0.1 F LiBr in water-methanol mixtures at 25 " C changes by about 20%. This lack of agreement with Stokes-Einstein behavior for methanol casts some doubt on the agreement in ethanol, for the latter may be due to fortuitous cancellation of other effects. However, the point remains that these solvent mixtures are not regular solutions, that the viscosity-composition diagrams have pro-
fP 7
1 1.3
'dc 'nP
3,
1.1 W
2,
0 v)
'U
v;
7
0.9
;
- 0.7 Vlv
%
Ethanol
Figure 4. Effect of the volume fraction of ethanol on the wave or peak height and half-wave or peak potentials for Knitrosoproline 1.00 X
MNOPro:
Y:
2 mV sec-'; LE -50 mV. Symbols as in Figure 3
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2415
Table IV. Various Supporting Electrolytes for Determination of N-Nitrosaminesa Supporting electrolyte
0.1 M LiCl + B-R (pH 1.8-1.9) 0.1 M HCl (pH 0.80) 0.1 M H,SO, + 0.1 M Na,SO, (pH 1.15) 0.1 M H,SO, + 0.1 M MgSO, -~ (pH 1 . i 2 ) 0.1 M H,SO, + 0.1 M CaSO, (PH 0.95) 0.1-M H,SO, + 1.0 M
E,,, V vs. SCE
-0.765 -0.675 -0.705
Concn range, IW (x 10’)
-AE, mV
100 50 and 100 50
-0.685
7-50 0.8-80 1-20
50
background current unstable
50
1-10
-0.70 2-6 50 (NH,),SO, (PH 1.05) 0.1M H,SO, + ( 1 0 - 2 -10-3) no peak appears; background 50 and 100 M Bu,NBr current small 0.1 M H,SO, + (10-2-10-3) -0.680 50 and 100 0.9-20 M Me,NCl (pH 1.1) 50 0.1 M H,SO, + 0.1 M -0.69 background current unstable Me,NCl 50 0.1M H,SO, + 10-3 M background current unstable Pr,NBr (pH 1.1) 0.1 M H,SO, + lo-’ M -0.840 50 1-8 Pr,NBr (pH 1.1) 0.1 M H2S0, + 5 x lo-, M -0.710 50 and 100 1-8 Et,NClO, (pH 1.2) Data for N-nitrosoproline. The other compounds give similar results.
nounced maxima, and that because of this, apparently trivial changes in solvent composition can cause substantial changes in current. Although we find a decrease in current in these mixtures, under slightly different conditions increases ir. current in going from aqueous to mixed solvents with alcohol or acetone also have been reported (21). The D P peak current is more affected by changes in solvent composition than are the dc or N P limiting currents. This is not surprising, because the rate determining step for the reduction involves reactant adsorption. Methanol 21 rid ethanol compete with the reactant for adsorption sites more effectively than does water. This could decrease the reaction rate which in turn would decrease the D P peak current. 40
0.88
30
0.88
Y
51 0.84 5
%- 20
-
P
I
0.82
10
0
J
L
‘
0.80
Figure 5. Effect of modulation amplitude on the peak height and peak potential for Knitrosopyrrolidine in 0.1 M LiCl containing 50 v/v
% B-R buffer at pH 1.94 1.08 X I O-, M NOPyr; Y: 2 mV sec-’
2416
-
-AE
0.1 M H,SO, + 0.1 M Me,NCl 0.1 M H,SO, + l o w 3M Pr,NBr (pH 1.1) 0.1 M H,SO, + lo-, M Pr,NBr (pH 1.1)
Presence of Surfactants. Because adsorption is important to the reduction rate, we have studied the effect of various surfactants on the D P current. We have investigated the effects of peptone (an enzyme digest of proteins), gelatin, methyl red, and Triton X-100. These compounds represent reasonable but simplified models of a complex matrix of surface active compounds. None of these substances affected the D P peak current a t concentrations S10-3%, although all decreased the current at higher concentrations. Effect of Modulation Amplitude. In general, the larger the pulse amplitude, the greater the peak current and the greater the sensitivity. Small pulse amplitudes, however, give better resolution and less instrumental error (14). For reversible reactions, the peak height-pulse amplitude relationship predicts that, at small pulse amplitudes, the peak current is linear in pulse amplitude but approaches as a limiting value the N P diffusion current. Therefore there is no advantage in going to very large pulse amplitudes (>50-100 mV, depending on n ) because there is not an appreciable gain in sensitivity. However, as one would expect, for an irreversible reaction the linear region of peak height dependence on pulse amplitude extends to much larger pulse amplitudes. This is illustrated in Figure 5 . This makes it much more advantageous to work at larger pulse amplitudes to obtain better sensitivity when one is dealing with irreversible reactions. Figure 5 also shows that, as in the reversible case, the peak potential is linear in pulse amplitude. However, the slope, ~ 0 . 4 is , less than the reversible slope of 0.5. Supporting Electrolyte and Detection Limit. For reduction of organic molecules with involvement of prior chemical reaction and adsorption the choice and concentration of supporting electrolyte can substantially change the reversibility and hence the magnitude of the D P peak current for the reaction. Cation adsorption can be expected to lower the reaction rate by competing with reactant adsorption and by reducing the proton activity in the reaction layer. It also, however, can improve the signal by reducing the differential capacity and hence the D P background current in the potential range of interest. We have examined several supporting electrolytes at about pH 1 where there is no variation in current with pH (cf. Figures 1-3). The re-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
I
i
I
&zy-supporting electrolytes r
0
1
ii t’ supporting electrolytes
0
0.2
0
0.6
0.8
1.0
0.8
1.0
1.2 -E
1.4
SCE
VI.
Flgure 6. Effect of tetrabutylammonium bromide on the peak for K nitrosoproline reduction
2.00 X
M NOPro; supporting electrolyte: 0.1 M H2S04 MTBAB (pH 1.0); u:2 rnV sec-’; A E -50 rnV
1.4
1.2 -E
0.6
0.4
0.4
0.2
VS.
SCE
Figure 8. Differential pulse polarograms of Knitrosoproline in 0.1 M M TMAC (pH 1.1) H2S04 containing MNOPro; u : 2 rnV sec-’; AE -50 rnV
(2.Od--12.0) X
0.20 I
-
+ 1.00 X
0.15
-
4 0.10 -
0.05
0
0.2
0.4
0.6
0.8
1.2
1.0 -E
Y S .
1.4
-
o:,
SC i
2
4,
Figure 7. Differential pulse polarograms of Knitrosoproline in 0.1 M HCI (pH 1.0) (1.00
- 20.0) X
8
6
I NOProl, M
(x
lo71
io
i2
Figure 9. Calibration curve Knitrosoproline by DP Supporting electrolyte: 0.1 M H2S04 4- 0.1 M Na2S04 (pH 1.15); u: 2 rnV peptone sec-’; AE -50 rnV; (0)without peptone; (A)
MNOPro; u: 2 rnV sec-l; A E -50 rnV
sults are summarized in Table IV. Especially in 0.1 M H 2 S 0 4 containing M tetrabutylammonium bromide, the D P background current is very small as shown in Figure 6 because of the adsorption of tetrabutylammonium ion. However, the reduction of N-nitrosamines is inhibited by the adsorption of these ions, and no peak appears. The data of Table IV for 0.1 M plus various tetralkylammonium salts illustrate the effects of the identity and concentration of supporting electrolyte on the rate of nitrosamine reduction. Concentration effects are shown by comparing results for MedNCl or Pr4NBr at different concentrations while effects of the nature of the cation are shown by comparing results in the 0.01 M range for Me4NC1, Et4NC104, Pr4NBr, and Bu4NBr. These results are in general accord with the discussion by Delahay (29). The effects of ionic strength were investigated from I = 0.01-0.51 M using solutions 0.01 or 0.1 M in HCl with
added KC1. In the D P mode, peak currents are constant for ionic strength I 1 0.04 M. At lower ionic strengths, the current increases with decreasing ionic strength as one might anticipate from double layer effects. Figures 7 and 8 show typical differential pulse polaroM level of NOPro in 0.1 M HCl or 0.1 M grams a t the H2S04 M tetramethylammonium chloride. If the current offset in the instrument and modulation amplitude of -100 mV are used, detection limits are about 8 X
+
M. For example, .calculation of the detection limit for NOPro under the conditions of Figure 9 according to the procedure of Skogerboe and Grant (30) gives dl = ts/m = 7.11 X M, where t is the t statistic, at the 99% confidence level, s the pooled standard deviation of the calibration curve (2.93 nA) and m its slope (0.1386 pA/pM). The
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2417
data from which this value was calculated are displayed in the calibration curve of Figure 9. The detection limit can also be given by dl = 3Sb/m where S b is the standard deviation of the background current. From background measurements at different times and in different supporting electrolytes, S b was estimated to be 3.7 nA, which gives dl = 8.1 X M . Reasonable agreement of these two methods permits some confidence that the calculated detection limit is realistic. We can assume measurement of current due to the analyte has a precision at least as good as that of the background determination. At concentrations greater than about ten times the detection limit, this inherent precision is generally better than the overall precision of determination in a practical sample. The detection limit for NOHOPro is somewhat higher than for the other two compounds. For NOPro, a detection limit of 8 X M corresponds to about 10 11811. which is adequate for laboratory studies and for many investigations of the prevalence of nitrosamines in the environment (31).
LITERATURE CITED (1) P. N. Magee and J. M. Barnes, Adv. CancerRes., I O , 163 (1967). (2) H. Druckrey, R. Preussmann, S. Ivankovic, and D. Schmahl, Z.Krebsforsch., 69, 103 (1967). (3) A. Wolff and A. E. Wasserman. Science, 177, 15 (1972). (4) T. Aune, Nord. Veterinaermed.,24, 356 (1972). (5) B. Goweniock and W. Luttke, Quart. Rev., 12, 321 (1958). (6) R. H. White, D. C. Havery, E. L. Roseboro, and T. Fazio. J . Assoc. Off. Anal. Chem., 57, 1380 (1974). (7) E. T. Huxel, R. A. Scanlan. and L. M. Libbey. J. Agric. Food Chem., 22, 698 (1974). (8) T. A. Gough and K. S.Webb, J . Chromatogr., 79, 57 (1973). (9) D. D. Bills, K. I. Hildum, R. A. Scanlan, and L. M. Libbey, J. Agric. Food Chem., 21, 876 (1973).
(10) E. T. Huxel, R. A. Scanian, and L. M. Libbey, J . Agric. FoodChem., 22, 698 (1974). (11) C. L. Walters, E. M. Johnson, and N. Ray, Analyst (London), 95, 485 (1970). (12) F. L. English, Anal. Chem., 23, 344(1951). (13) J. G. Osteryoung and R. A. Osteryoung, Am. Lab., 4&(July), 8 (1972). (14) J. H. Christie, J. G. Osteryoung, and R. A. Osteryoung, Anal. Chem., 45, 210 (1973). (15) I. M. Kolthoff and A. Liberti, J. Am. Chem. SOC.,70, 1884 (1948). (16) B. Martin and M. Tashdjian. J . Phys. Chem., 60, 1028 (1956). (17) H. Lund, Acta Chem. Scand., 11, 990(1957). (18) L. Holleck and R. Schindler, Z.Elektrochern., 62, 942 (1958). (19) F. Pulidori. G. Borghesani, C. Bighi, and R. Pedriali, J . Electroanal. Chern.,27, 385 (1970). (20) G. Borghesani, F. Pulidori, R. Pedriali, and C. Bighi, J . Nectroanal. Chem.,-32,303 (1971). (21) R. Zahradnik. E. Svatek, and M. Chvapil, Collect. Czech. Chem. Commun.. 24. 347 119.59). , (22) D.J. Myers and Janet Osteryoung, Anal. Chem., 46, 356 (1974). (23) J. H. Christie and R. A. Osteryoung, J. Nectroanal. Chem., 49, 301 (1974). (24) A. E. Wasserman, Research Leader, Meat Composition and Quality Research, USDA Eastern Regional Research Laboratory, private communication. (25) W. Lijinsky, L. Keefer, and J. Loo, Tetrahedron, 26, 5137 (1970). (26) I. M. Kolthoff and J. J. Lingane, "Polarography", Vol. 1, 2nd ed., Interscience, New York, N.Y., 1952, p 86. (27) Ref. 26, p 52. (28) H. C. Jones, "Conductivity and Viscosity in Mixed Solvents". Carnegie Institution of Washington, Publ. No. 80, (1907). (29) P. Deiahay, "Double Layer and Electrode Kinetics", Interscience, New York, N.Y., 1965, p 297. (30) R . K. Skogerboe and C. L. Grant, Spectrosc. Lett., 3 , 215 (1970). (31) R. Preussmann, "On the Significance of KNitroso Compounds as Carcinogens and on Problems Related to Their Chemical Analysis", pp 6-9, in NNitroso Compounds Analysis and Formation, IARC Sci. Publ. No. 3, Lyon (1972). I~
.~
~
~~
RECEIVEDfor review July 3, 1975. Accepted September 5, 1975. This work was supported in part by NIH Grant CA 15028-01 and by NSF Grant GP 31491X.
Spectrophotometric Determination of Methyl Glyoxal with 2,4Dinitrophenylhydrazine Robert P. Gilbert and Richard B. Brandt' Department of Biochemistry, Virginia Commonwealth University, Medical College of Virginia, MCV Station-Box
A sensitive method has been developed for the spectrophotometric determination of methyl glyoxal (MeG) uslng an ethanol-HCI solution of 2,4-dinitrophenylhydrazlne (2,4DNPH). Optimal~conditlonsInclude: 2 X 10-4M 2,4-DNPH in 12 ml of concentrated HCI per 100 ml of ethanol, heated 40 min at 42 k 1 OC showed over 99% of reaction completion with MeG when measured at the absorption maximum at 432 nm. The system conforms to Beer's law up to 1.38 X 10-5M. A molar absorptivity of 3.36 X l o 4 cm2/mmol was found. Glutathione, o,L-lactate, pyruvate, and glucose did not interfere with the assay at expected biological levels. Yeast glyoxalase I activlty was measured and found to correspond to the actlvity determined by a standard method. The method will have application to measurement of glyoxalase I activity in tissues.
Methyl glyoxal (MeG), also known as pyruvaldehyde, is an a,j3 dicarbonyl. At one time in the history of biochemistry, it was thought to be the source of lactic acid from the glycolytic pathway ( I ) . MeG is the substrate for the combined enzyme system composed of glyoxalase I and glyoxal
A u t h o r t o w h o m correspondence should be addressed.
2418
727, Richmond, Va. 2 3 2 9 8
lase I1 with glutathione as a cofactor and D-lactate as the product of catalysis. Glyoxalase I is S-lactoyl glutathione methylglyoxal-lyase (isomerizing), E.C. 4.4.1.5, which in the presence of reduced glutathione (GSH) converts MeG to a hemimercaptal, S-lactoyl glutathione. The second enzyme, glyoxalase I1 is S-2 hydroxyacylglutathionehydroxylase, E.C. 3.1.2.6, which catalyzes the conversion of S-lactoyl glutathione to D-lactate, rather than L-lactate, the usual product of glycolysis. This is an enzyme system found in many varied tissues and organisms while its specific function is unknown. Interest in the enzyme system and the substrate have been stimulated by the finding of SzentGyorgi ( 2 ) who suggested its involvement in inhibition of growth. His observations have led to a number of publications by others on the glyoxalase system. The analysis of MeG has been performed by manometric ( 3 ) ,arsenophosphate colorimetric ( 4 ) , oxidative-titrimetric (5), isotope dilution ( 6 ) , and UV spectrophotometric (7, 8) procedures. The use of a visible spectrophotometric method using 2,4-dinitrophenylhydrazine(2,4-DNPH) has been reported (9, I O ) , but we found the procedures not applicable for multiple analysis of glyoxalase I in biological systems. We report here a spectrophotometric procedure for quantitative assay of MeG applicable to the measurement
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
