1782
Anal. Chem. 1983, 55, 1782-1786
Liquid Chromatographic Determination of Hydrazines with Electrochemically Pretreated Glassy Carbon Electrodes K. Ravichandran and Richard P. Baldwin* Department of Chemistry, University of Louisville, Louisville, Kentucky 40292
Electrochemical pretreatment of glassy carbon electrodes was shown to enhance significantly the analytical capabllltles of the electrodes for the determination of simple hydrazines. The primary effect of the initial application of brief anodic and cathodic potentials was a shift of the oxidatlon waves observed for hydrazlne and its monomethyl and dimethyl derivatlves to potentials 0.2-0.8 V lower than those required at untreated glassy carbon surfaces. When employed as amperometric sensors followlng llquld chromatographlc separation of the hydrazines, detection limits from 2 to 50 pmol inJectedwere obtained with an applied potential of 4-0.50 V vs. Ag/AgCI. At higher hydrarlne concentrations, sufflclentiy selective response was obtained at detector potentials as low as +0.10 V that no sample treatment was required for quantitation of 1,l-dimethylhydrazine In urine at the 125-pmol level.
Hydrazine compounds represent an important family of organics whose wide use in a number of industrial and pharmacological applications and documented carcinogenic behavior in laboratory animals make their detection and quantitation problems of considerable analytical interest. As a consequence, recent activity has been directed toward the development of sensitive and selective analytical methods for the determination of hydrazines in a variety of sample matrices. Because of the often complex nature of the sample involved and the frequent need to resolve simultaneously several closely related hydrazine species, gas or liquid chromatographic procedures have most commonly been suggested. Due to the relatively modest absorptivities of most simple hydrazines in practically accessible wavelength regions, acceptable sensitivity by use of spectrometric monitoring has been achieved only after preliminary derivatization processes have been carried out. Most commonly, these procedures have involved coupling of the hydrazine moiety with aldehydes such as nitro-2-hydroxybenzaldehyde ( I ) , pentafluorobenzaldehyde (2), and salicylaldehyde (3) and have yielded liquid chromatographic detection limits typically ranging from 1 to 3 nmol injected onto the column for hydrazine and its mono- and dimethyl derivatives (3). In a recently reported procedure applicable to some commercial pharmaceutical preparations, Butterfield et al. ( 4 , 5 )were able to determine hydrazine itself a t the 3-pmol level after precolumn formation of the benzaldehyde derivative; however, the detection of other hydrazines was not attempted. Most recently, the use of electrochemical detection techniques have begun to be examined for hydrazine determination following liquid chromatography (LCEC). Such approaches employing the direct oxidation of the hydrazine functionality have been limited by the fact that most simple hydrazines undergo oxidation at conventional electrode surfaces only a t a substantial overpotential. Thus, Fiala and Kulakis (6),employing LCEC at a glassy carbon electrode maintained at +1.0 V vs. Ag/AgCl, were able to achieve
sensitivities only slightly improved over those obtained via salicylaldehyde derivatization/UV detection and significantly poorer than the sensitivity usually characteristic of the amperometric detection approach. Furthermore, detector selectivity which, in LCEC, is directly determined by the applied electrode potential would have been significantly enhanced by the use of less extreme working electrode potentials. In an attempt to circumvent this problem, Kester and Danielson (7) applied LCEC to the salicylaldehyde derivatives themselves which, because of their constituent phenolic group, were expected to be somewhat more easily oxidized than the parent hydrazines. As a result, the potential required for detection was decreased to +0.80 V; and detection limits were slightly improved. Considerable work has been directed toward the enhancement of electrode response toward many activation-controlled redox processes (8-14). In this work, both specific chemical modification of the conventional electrode surface by the attachment of appropriately selected electrocatalysts and general electrode conditioning by chemical or electrochemical oxidation of the native surface have been shown to be effective in producing a more ideal electrode response. Numerous examples of the latter approach have been reported, most of which have utilized the application of alternating oxidizing and reducing potential steps of varying magnitude and duration to either glassy carbon or pyrolytic graphite electrode substrates (10-14). In particular, Engstrom (14)has described a simple electrochemical pretreatment procedure for glassy carbon which produced a substantial decrease in the potential of the voltammetric wave observed for the oxidation of hydrazine (and other analytes as well). In this paper, we will examine in detail the effect of this electrochemical pretreatment on the voltammetric oxidation of several hydrazine derivatives and will demonstrate the use of these electrodes to facilitate the amperometric electrochemical detection of these species following liquid chromatography. These simple electrode conditioning procedures served to greatly increase the sensitivity of LCEC for simple hydrazines and also permitted operation at significantly lower detector potentials with no prior sample treatment or derivatization steps.
EXPERIMENTAL SECTION Reagents. Hydrazine solutions were prepared from hydrazine sulfate (Sigma Chemical Co.). Methylhydrazine, 1,2-dimethylhydrazine (1,2-DMH),and 1,l-dimethylhydrazine(1,l-DMH)were purchased from Aldrich. All were obtained in reagent grade and were used without further purification. All solutions were prepared with deionized water. Voltammetry. Cyclic voltammetry experiments and electrode pretreatment procedures were performed with a Bioanalytical Systems (West Lafayette, IN) Model CV-1B potentiostat and glassy carbon working electrode. Before conditioning, the electrode was polished three times with an alumina slurry. The duration of each polishing was 1 min, and the electrodes were rinsed thoroughly with deionized water each time. After the final polishing, the electrodes were also rinsed with acetone and allowed to air-dry. A three-electrode cell configuration with a standard calomel reference electrode and a platinum wire counterelectrode was
0003-2700/83/0355-1782$01.50/00 1883 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
employed for all voltammetric experiments. The supporting electrolyte/buffer consisted of 0.10 M KN03 and 0.010 M NazHP04 which was adjusted to pH 7 with 0.015 M HN03. Chromatography. The liquid chromatograph employed was a modular system consisting of a Waters Asi3ociates (Milford,MA) Model M-6000 pump, a Rheodyne (Berkeley, CA) Model 71 25 sample injector with a 20-pL sample loop, and a Bioanalytical Systems Model LC-3 or LC-4A amperometric detector equipped with a Model TL-5 thin-layer glassy carbon electrode assembly. Electrode conditioning for the LCEC experiments was accomplished by means of the same procedure found effective in the cyclic voltammetry experiments. The reference electrode was a Ag/AgCl electrode. All separations were performed on a 30-cm, 10-pm octadecylsilane column (Regis Chemical Co., Morton Grove, IL). Unless specified otherwise,the mobile phase consisted of the same pH 7 KN03/Na2HP04solution described above for the electrochemical studies.
RESULTS AND DISCUSSION Voltammetry of Hydrazines at Prebtreated Electrodes. A variety of electrochemical treatment procedures for glassy carbon involving application of both anodic and cathodic potentials for different time periods was investigated by Engstrom (14). The most effective conditioning sequence for hydrazine was found to consist of a 5-min preanodization a t +1.75 V vs. SCE followed by a 10-s precathodization at -1.2 V. In this work, the identical electrode pretreatment sequence was employed for all experiments. Only one difference should be noted. In Engstronn’s work, the pretreatment was carried out with the electrode immersed in a solution containing an appropriate concentration of hydrazine (or any other analyte under investigation). During the cortditioning procedure employed here, however, the glassy oarbon surface was in contact with a solution containing only buffer/electrolyte. No significant differences in subsequent electrode behavior were observed as a result of this change. The cyclic voltammlograms observed for a hydrazine-containing solution a t botlh untreated and pretreated electrode surfaces are shown in Figure 1A. In the former case, the oxidation consisted of a broad, irreversible wave having a peak potential of +0.98 V vs. SCE. With the pretreated glassy carbon surface, the anodic wave remained broad and irreversible but was shifted down to +0.2:! V. Electrodes electrochemically conditioned in this manner continued to exhibit this improved response toward hydrazine for several hours whether immersed at open circuit in blank solution or cycled continuously in the pyesence of hydrazine. The only significant deterioration in response consisted of a gradual shift of the hydrazine redox wave back to more positive potentials. This shift was consistently observed to take place beginning during the first hours of the treated electrodes’ use and amounting to approximately 100 mV after 25-30 h. Repolishing of the treated electrode surface with alumina effectively restored the electrode response toward hydrazine to its original nonideal state. Thew observations agree well with results reported previously by Engstrom (14). The same electrode pretreatment procedure was also found to be effective in decreasing the potential required for the oxidation of the monomethyl- and the symmetrical and unsymmetrical dimethylhydrazine species as well. The voltammograms obtained for these compounds1 are also summarized in Figure 1. At the untreated electrode surface, only the 1,l-DMH derivative yielded a well-formed, though still irreversible anodic wave at a comparatively low potential. But, even in this case, pretreatment of the glassy carbon still produced a significant lowering of the oxidation potential required for the analyte. The one deleterious effect produced as a result of the preanodization and precathodization steps consisted of a marked increase in the background current level observed for
1783
2 0 pA
20 pA
20 FA
J + 1.0
I
1
+O 6
+0.2
1
- 0.2
POTENTIAL V v s S C E
Flgure 1. Cyclic voltammograms at untreated (- - -) and electrochemically pretreated (-) glassy carbon electrodes of (A) 1.7 X M hydrazine, (E) 1.9 X lo-’ M, methylhydrazine, (C) 8.3 X M 1,P-DMH, and (U) 1.3 X lo-* M 1,l-DMH. Potential scan rate was 10
mV/s.
the treated electrode surfaces. The exact magnitude of the background varied, of course, depending on the specific experimental conditions in effect. However, in all cases, use of the electrochemically pretreated electrodes resulted in a rough doubling of the background current over that found for the unmodified surface. LCEC at Pretreated Electrodes. Chromatograms obtained for a mixture of the four hydrazine derivatives using LCEC a t both untreated and pretreated glassy carbon electrodes are provided in Figure 2. In both cases, the electrode potential was maintained at +0.10 V vs. Ag/AgCl; and the same hydrazine mixture was employed for each chromatogram. At the extremely low potential used here, a large response was obtained for the electrodes which had undergone the electrochemical conditioning sequence even though no signals at all could be detected for the hydrazines at the conventional electrode. This behavior was exactly that expected on the basis of the cyclic voltammograms obtained above for the similar electrode treatment strategies. In fact, the peak currents observed for the hydrazines at the untreated surfaces were found to match the current levels found for the preanodized and -cathodized electrodes only when the electrode
1784
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
Table I. Comparison of Detection Limits detection limit, pmol LCEC LCEC' (no pretreatment) (with pretreatment)
LC-uv (254 nm)" hydrazine methylhydrazine 1,2-dimetliylhydrazine 1 , l-dimethylhydrazine a Salicylaldehyde derivatives; see ref 3. AgCl; this work.
1250 2200
2500 370 170 120
2200
3.1
2.2 50 13
E = t 1.0 V vs. Ag/AgCl, no derivatization; see ref 6.
I
E = t 0.50 V vs. Ag/
E
A
i
b
P
x /x
I
!+:
L;,
2 '
+0.2
X
Y
+0,6
x'
t1!0
+0.2
POTENTIAL V vs
,,*,x',
et' t0.6
t1.0
A~/A~CI
Hydrodynamic voltammograms at untreated ( X ) and electrochemlcally pretreated (0)glassy carbon electrodes for (A) hydrazine, (B) methylhydrazine, (C) l,ZDMH, and (D) 1,l-DMH. All concentrations were the same as those given in Figure 2. Flgure 3.
Flgure 2. Chromatograms of hydrazine mixture at 4-0.10 V vs. Ag/ AgCl at (A) untreated and (B) electrochemically pretreated glassy carbon electrodes. Peaks correspond to (1) hydrazine 8.4 X M, (2) methylhydrazine 9.6 X M, (3) 1,P-DMH 3.5 X M, and (4) 1,l-DMH 1.3 X M. Flow rate was 2.5 mL/min.
potential was maintained some 0.5-1.0 V higher. The hydrodynamic voltammograms, that is, peak current vs. potential profiles, for each of the four hydrazines at treated and untreated electrodes are shown in Figure 3. For the untreated electrodes, optimum response in terms of the current levels obtained occurred a t potentials greater than +1.0 V. This behavior clearly accounts for the less than ideal analytical results achieved previously for LCEC of hydrazines at these types of surfaces. Pretreated glassy carbon yielded hydrodynamic voltammograms which exhibited well-defined plateaus a t potentials in the vicinity of +0.50 V vs. AgIAgC1 for all four hydrazines. Optimum LCEC response for these electrodes should thus occur (as observed above) a t significantly reduced potentials compared to the untreated surfaces. The only difference was found for the 1,l-DMH which exhibited, in addition, another plateau a t somewhat higher potentials. The reason for this second wave is not entirely clear. However, of the hydrazines examined here, only this compound was found to exhibit a well-defined redox wave on its own at relatively modest potentials at the untreated glassy carbon surface. In terms of sensitivity, the optimum LCEC response should be obtained for the pretreated electrode a t a potential of roughly +0.5 V vs. SCE for all of the hydrazine species except
perhaps the unsymmetrical dimethyl species. Accordingly, this potential was selected in order to obtain calibration curves and detection limits for each of the compounds. Calibration curves so obtained were linear over the entire range of concentrations examined, possessing correlation coefficients greater than 0.99. The highest concentrations employed were the following: hydrazine, 2.5 ppm; methylhydrazine, 2.6 ppm; 1,2-DMH, 30 ppm; and l,l-DMH, 16 ppm. Reproducibility of repeated injections as evidenced by their relative standard deviation was typically less than 5%. At the nanomole injection level and for 10 injections, this value for hydrazine was 4.9%and for methylhydrazine was 2.1%. The detection limits determined for the compounds at the pretreated electrodes ranged from 2 to 50 pmol or from 10 to 800 times lower than those previously reported by using either UV absorption of the salicylaldehyde derivatives at 254 nm or electrochemical oxidation at untreated glassy carbon electrodes at +1.0 V vs. SCE. These results are summarized in Table I. As expected from the cyclic voltammetry described above, the activity of the electrochemically treated electrodes for LCEC was observed to decrease slightly with time. Thus, for hydrazine and monomethylhydrazine, the slope of the calibration curve obtained decreased by some 10% during the course of 10-12 h of continuous chromatography. No effect was observed for either of the dimethyl species over the same time period. Ordinarily, pretreated electrodes continued to
ANALYTICAL CHEMISTRY, VOL. 55, NO. 11, SEPTEMBER 1983
IOnA
i’.
1,l -DMH
1785
nA
B
A
1,l-DMH
;i
B
I
J t, min.
t,min.
Flgure 5. Chromatograms obtained at +0.10 V vs. Ag/AgCi for (A) urine blank and (9) same urine sample doped with 7.0 ppm 1,l-DMH. Conditions are described in the text.
expect to achieve complete resolution of the analyte peak from all others. Clearly, in lieu of relying on elaborate and usually time-intensive sample treatment procedures carried out prior to the LCEC measurement, the latter selectivity approachthat is, the ability to use a very modest working potentialrepresents an attractive analysis feature. I With this point ofview in mind, the suitability of electro- -0 0 8 16 chemically pretreated glassy carbon electrodes for the det, rnin. t, min. termination of hydrazines in actual physiological matrices was Figure 4. Chromatogranis obtained at +0.50 V vs. Ag/AgCi for (A) examined. For this purpose, the determination of 1,l-DMH urine blank and (9) same urine sample doped with 7.0 ppm 1,l-DMH. in doped urine samples was selected as a test system. The Conditions are described in text. chromatograms obtained for such a sample at both +0.50 V and +0.10 V vs. Ag/AgCl are shown in Figures 4 and 5, reshow significantly enhanced response on the second day of spectively. In both cases, a mobile phase consisting of 5% use and beyond. However, in view of the simple and rapid CH3CN/95% aqueous phosphate buffer was employed a t a electrode polishing and electrochemical conditioning steps flow rate of 0.8 mL/min; and the only prechromatographic required, it is recommlended that pretreatment be performed sample treatment consisted of passing the doped urine samples daily prior to each day’s work. through a 3-km glass filter prior to injection. As seen in Figure In practical analytical applications, the use of organic 4,the chromatographic response obtained at +0.50 V for a modifiers in the mobile phase would very likely be required typical urine blank (curve A) and for the same sample doped to aid in the chromatographic separation. Consequently, the effect of moderate amounts of organic solvents on the perwith 1,l-DMH a t a level of 7.0 ppm (curve B) clearly shows formance of electrochemically pretreate!d electrodes was also that the hydrazine peak appears in a region containing sizable, overlapping background signals. However, upon decreasing examined. As the two modifiers of greatest interest in LCEC are methanol and acetonitrile, binary mobile phases containing the potential of the pretreated electrode to +0.10 V (Figure each of these solventia were employed. During continuous 5), the background is drastically diminished and the 1,l-DMH day-long chromatograplhic runs using either 15% CH30H/H20 peak was completely resolved. As expected from the hydrodynamic voltammogram previously obtained for the analyte, or 10% CH3CN/H20,no deterioration in response was obsubstantially less current was obtained at the lower potential, served for the electrochemically pretreated electrodes. I t is but the sharp decrease in background clearly makes this the anticipated that higher organic fractions could be employed potential of choice for direct quantitation of 1,l-DMH in urine. without difficulty. For applications involving the determination of analytes In fact, a t this potential, no significant background peaks present in real samples, it is oftentimes the selectivity of the corresponding to strongly retained components (i.e., retention analysis approach which determines its practicality and real time greater than 12 min) of the urine blank were observed. sensitivity. In LCEC, such selectivity is determined both by The detection limit estimated for 1,l-DMH in urine by direct the degree to which the chromatography is able to isolate the injection a t +0.10 V was less than 125 pmol. Even with no compound of interest from other components of the sample pretreatment of urine samples other than filtering, no sigmatrix and by the degree of specificity exhibited by the nificant electrode passivation was observed to take place electrochemical detecitor-which in turn is related primarily during the course of 10-12 of continuous chromatography. Electrochemical pretreatment of conventional electrode to the electrode potential needed to achieve acceptable quantitation. In a complex sample, it 11soften unrealistic to surfaces represents an experimentally convenient and, as
Anal. Chem. 1983, 55, 1786-1791
1786
shown in this work, analytically useful surface modification strategy. The method appears to be much less specific than other commonly suggested modification approaches and results in an electrode surface whose actual chemical composition has been only incompletely characterized to this point in time. However, the electrochemically pretreated glassy carbon surface possesses suitable characteristics in terms of stability and reproducibility for direct application in LCEC. The hydrazines represent one class of anaytes for which both the sensitivity and the selectivity of the chromatographic determination can be dramatically increased. With the pretreated electrodes, moderate working electrode potentials can be employed to determine numerous hydrazines with a sensitivity comparable to that typically observed for the LCEC of ideally oxidizable analytes. On the other hand, unusually selective detection following electrochemical pretreatment can also be achieved for the hydrazines examined of electrodes by utilization of potentials as low as +0.10 V vs. AgIAgC1. Applications of these electrodes in the LCEC of other activation-controlled oxidations appear promising. Registry No. C, 7440-44-0; 1,2-DMH, 540-73-8; l,l-DMH,
57-14-7; hydrazine, 302-01-2; methylhydrazine, 60-34-4.
LITERATURE CITED (1) Neurath, G.; Luttich, W. J . Chromatogr. 1968, 3 4 , 257-258. (2) Llu, Y.-Y.; Schmeltz, I.; Hoffmann, D. Anal. Chem. 1974, 4 6 , 885-889. (3) Abdou, H. M.; Medwick, T.; Bailey, L. C. Anal. Chim. Acta 1977, 9 3 , 221-226. (4) Butterfield, A. G.; Curran, N. M.; Lovering, E. G.; Matsui, F.: Robertson, D. L; Sears, R. W. Can. J . Pharm. Scl. 1981, 16, 15-19. (5) Matsui, F.; Butterfield, A. G; Curran, N. M; Lovering, E. G.; Sears, R. W.; Robertson, D. L. Can. J . Pharm. Sci. 1981, 16, 20-22. (6) Fiala, E. S.;Kulakis, C. J . Chromatogr. 1981, 214, 229-233. (7) Kester, P. E.; Danielson, N. D. 1983 Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, March 1983;Abstract No. 172. (8) Snell, K. D.; Keenan, A. G. Chem. Soc. Rev. 1979, 8 , 259-282. (9) Murray, R. W. Acc. Chern. Res. 1980, 13, 135-141. (IO) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1975, 4 7 , 1337-1343. (11) Blaedel, W. J.; Mabbott, G. A. Anal. Chem. 1978, 5 0 , 933-936. (12) Wightman, R. M.; Paik, E. C.; Borman, S.; Dayton, M. A. Anal. Chem. 1978, 50, 1410-1414. (13) Gonon, F. G.; Fombarlet, C. M.; Buda, M. J.; Pujoi, J. F. Anal. Chem. 1981, 53, 1386-1389. (14) Engstrom, R. C.Anal. Chern. 1982, 5 4 , 2310-2314.
RECEIVED for review April 21, 1983. Accepted June 20, 1983.
Liquid Chromatographic Determination of Amino and Imino Acids and Thiols by Postcolumn Derivatization with 4-Fluoro-7-nitrobenzo-2,1,3-oxadiazole Yoshihiko Watanabe and Kazuhiro Imai*
Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
A new postcolumn derlvatlratlon method wlth use of 4fluoro-7-nltrobenro-2,1,3-oxadlazole (NBD-F) for highly and sensltlve determination of amino and lmlno aclds and thiols has been achieved. These compounds were automatically separated by cation exchange resin, reacted with the reagent to produce highly fluorescent NBD derlvatlves, and detected wlth a spectrofluorometer (A,, = 470 nm, A, = 530 nm for amlno and Imino aclds; A,, = 450 nm, A,, = 520 nm for thlols). As little as 5, 50, and 10 pmol of Pro, Tyr, and Cys can be detected, respectively. This method was applied to the determlnatlon of amlno and lmlno aclds In blood dlsks of normal and pathological newborns (phenylketonurla, maple syrup urine disease, and tyrosinosis). The values obtalned were compared wlth those obtalned by the o-phthalaldehyde (OPA) method.
to generate primary amines from the imino acids has been adopted. No facile way of detection of imino acids and other amino acids has yet been found to the best of our knowledge. A possible candidate is the recently developed fluorogenic reagent for amines, NBD-F (4-fluoro-7-nitrobenzo-2,1,3-oxadiazole) (10-12), which has the ability to react with both primary and secondary amines (Figure 1) under attainable reaction conditions using two or three solvent delivering pumps for buffer and reagents. In this paper, we report the postcolumn reaction and fluorometric detection of amino and imino acids with NBD-F after separation by ion exchange chromatography used with the high-performance amino acid analyzer. Some thiols, e.g., cysteine, homocysteine, and glutathione, are also quantitated by a modified detection system. The proposed method is applied to a profile analysis of amino and imino acids in blood samples on paper disks of 3 mm diameter for the diagnosis of inborn errors of metabolism.
In the past, ninhydrin has been used for the colorimetric determination of amino acids in biological fluids with an automatic analyzer (1). With the advent of high-performance liquid chromatography (HPLC) more efficient separations in shorter times have become possible (2,3). Recently, fluorometry has been introduced in the detection system to increase the sensitivity of the method (2-5). The fluorogenic reagents, fluorescamine (6) and o-phthalaldehyde (OPA) (7), are well suited for the sensitive detection of amino acids having primary amino groups; however, the imino acids, such as proline and hydroxyproline do not yield fluorescence with these reagents (2,3). Therefore, the addition of oxidizing reagents, such as N-chlorosuccinimide (8) or sodium hypochlorite (91,
EXPERIMENTAL SECTION was Reagents. NBD-F (4-fluoro-7-nitrobenzo-2,1,3-oxadiazole) synthesized by the method of Nunno et al. (13). Alloisoleucine (ICN-K&K Inc., New York, NY) was kindly donated by K. Suzuki of the University of Tokyo. All the other amino and imino acid standards were purchased from Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan). Amino and imino acid standard solution (2.5 fimol/mL) was purchased from Ajinomoto Co., Ltd. (Tokyo, Japan). Filter papers which were individually applied with standard amino acid solutions (0.5-20 mg/dL) of methionine, leucine, tyrosine, phenylalanine, and histidine were purchased from Fuji Zoki Pharmaceutical Co., Ltd. (Tokyo, Japan). A number of filter papers spotted with blood from normal and pathological newborns were kindly donated by H. Naruse
0003-2700/83/0355-178680 1.50/0 0 1983 American Chemlcal Society
