very useful in systems where users are charged for the CPU time they use in library searching. A library search system based on the method. outlined has been implemented in BASIC P-LUS on a PDP11/45.
ACKNOWLEDGMENT The author thanks J. K. MacLeod and C. MacDonald for their helpful comments and encouragement in this work and S. Heller and G . Milne for providing spectra from the EPA/ N I H data base.
CONCLUSION I t has been demonstrated that the series displacement index is a useful parameter for classifying mass spectra. T h e precision t h a t this method lacks relative t o some of the other methods for classification is compensated for by the fact that it provides a one-dimensional representation. There is considerable evidence to suggest that the series displacement index can provide valuable information about structural influences in a given class. Also SDI-structured library searching can lead too close to an order of magnitude reduction in t h e search space. Such a saving should enhance the effectiveness of large library search systems.
LITERATURE CITED (1) F. W. McLafferty,“Interpretation of Mass Spectra”, Benjamin, New York, N.Y., 1966. (2) D.H. Smith, Anal. Chem., 44, 536 (1972). (3) L. R. Crawford and J. D. Morrison, Anal. Chem., 40, 1469 (1968). (4) P. C. Jurs, Anal. Chem., 43, 22(1971). (5) E. von Sydow and R. Ryhage. Acta Chem. Scand., 17, 2025 (1963). (6) S. Grotch, Anal. Chem., 43, 1362 (1971). (7) H. S.Hertz, R . A. Hites, and K. Biemann, Anal. Chem., 43, 681 (1971). (8) S.R. Heller, Anal. Chem., 44, 1951 (1972).
RECEIVEDfor review March 11, 1976. Accepted June 1, 1976.
Direct Microdetermination of Manganese in Normal Serum and Cerebrospinal Fluid by Flameless Atomic Absorption Spectrophotometry Donald J. D’Amico Abraham School of Medicine, University of Illinois, Chicago, Ill. 606 16
Harold L. Klawans* Division of Neurology, Michael Reese Medical.Center, Chicago, 111. 606 16
The concentrations of manganese in normal human serum and cerebrospinal fluid as determined in 50-pl samples by flameless atomic absorption spectrophotometry are 1.02 f 0.19 ng/ml and 0.66 f 0.10 ng/ml, respectively. Instrumental parameters are extensively discussed. Possible sources of contamination and error are given.
Many intriguing questions have been raised concerning the biochemical and clinical importance of manganese (1, 2). These remain largely unanswered, as the difficulties inherent in assaying this trace element have precluded widespread investigation. As a result, the normal concentration of manganese in human serum is still subject to considerable debate, with a range of 0.57 t o 24 ng/ml having been reported in the literature (3-12). Normal values for cerebrospinal fluid (CSF) manganese have not been widely investigated (13). Most researchers have employed either neutron activation analysis or atomic absorption spectrophotometry. Neutron activation offers exquisite sensitivity for manganese (14),but the lengthy procedures and expensive instrumentation required are serious drawbacks when considered for present day routine application. Conventional flame atomic absorption spectrophotometry suffers from a lack of sensitivity (15),and concomitant extraction procedures add time and often manganese as well. The development of flameless atomic absorption, however, has dramatically lowered detection limits for manganese (16), with concentrations in most biological media clearly within its limits. The technique is rapid and
accurate and has gained widespread acceptance in elemental analysis. Perhaps its greatest advantage is its increasing availability since this instrument is rapidly becoming a standard clinical laboratory equipment item. The flameless apparatus employed in the present study contained a graphite cylinder which could be automatically heated through three successive stages: drying, charring, and atomization (during which an absorption signal is generated). The validity of the method required optimization and verification of the instrumental parameters for both serum and CSF; simultaneous use of a deuterium background corrector allowed samples to be analyzed directly without pretreatment or dilution.
EXPERIMENTAL Apparatus. The HGA-2100 (Perkin-Elmer)graphite furnace was used in conjunction with Perkin-Elmer Models 306 Atomic Absorption Spectrophotometer,56 Recorder, Deuterium Arc Background Corrector, and hollow-cathode lamps for manganese and lead, operated a t 18 and 10 mA, respectively. The graphite cylinder was purged with argon (zero grade, Liquid Carbonics)at a flow rate of 27 cm3/min. Samples were placed in the furnace with the desired Eppendorf pipet (10-50 111) fitted with disposable tips. Syringes and 20-G needles (Monoject)and snap-lock plastic tubes (Falcon)were used for serum collection: Travenol Lumbar Puncture Kits and snap-lock tubes were used for CSF collection. Reagents and Standards. Highly purified water was obtained by passing deionized water through a Brinkmann Quartz Bidistiller,and was used throughout. Standards (5.0 and 10.0 ng/ml) were prepared by appropriate dilutions from 1000 pglml manganese stock solutions (Fisher Scientific Co., Chicago, Ill., Catalog No. 50-M-81).
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
1469
Table I. HGA-2100 Operating Parameters for the Determination of Manganese in Human Serum and Cerebrospinal Fluid (CSF)a Char,
Dry, s / 1 2 5 "C
s/800 O C
120c 6 Od
60 60
Atomization, s/ Wavelength, 2300 "C nm
8 8
279.7 279.7
Slit width,b mm
Argon flow,
10 10
cm3/
m in
27 27
a Based o n a 50-1.11 sample. b Corresponds t o a spectral band width of 0.7 nm. C Serum. d CSF.
250
500
750
1250
1000
Temperature PC)
Table 11. Minimum Charring Parameters for Complete Background Correction with Deuterium Arca 25 p1
Figure 1. Effect of increasing charring parameters on the signal produced by 500 pg of manganese (as dilute HN03 standard) 0 - 0 10-s charring; 0 - 0 60-s charring
CSF :;I
Serum 60 si600 "C 60 si700 "C CSF 30 si600 "C 60 ~ 1 6 0 0 "C a Values determined at the Pb 280.2 nm line with simultaneous operation of deuterium arc t o yield A < 0.001.
SERUM
4
t
so p1
\\
n
~~
~
Table 111. Normal Manganese Concentrations in Human Serum and CSF Serum Number of samples: 19 Individual Mn levels (ngiml): 0.74, 0.77, 0.80, 0.81, 0.84, 0.85, 0.87, 0.'90, 0.93, 0.96, 1.04, 1.09, 1.17, 1.19, 1.19, 1.21, 1.24, 1.25, 1.25
Mean: 1.02 Standard deviation: 0.19 CSF Number of samples: 1 0 Individual Mn levels (ngiml): 0.55, 0.57, 0.60, 0.61,
.3
.2 .I
500
600 700 800 Temperature ( '0
500
600
700
Figure 2. Background signal prior to deuterium arc correction as a function of charring parameters 0 -0 25 plat 30 s; 0 - 0 25 plat 60 s; 0 - 0 50 pI at 30 s; W s; background was measured at the Pb 280.2 nm line
-
Mean: 0.66 Standard deviation: 0.10
50 plat 60
Procedure. Blood samples were obtained by venipuncture from 20 presumed healthy volunteers and were transferred to plastic tubes
and allowed to clot. Clear serum was obtained by centrifugationand immediately removed from the packed cell column. Samples of CSF from 10 subjects were assayed. In each case, an aliquot of the fluid had been shown to be normal in terms of protein, glucose, and chloride concentrations and cell count. After passing through the lumbar puncture kit described above, it was collected in plastic tubes. The sample (usually 50 pl) was introduced into the furnace and the sequential heating and analysis were performed. The specific operating parameters are given in Table I. The selection of these parameters is dealt with extensively in the discussion. All samples were analyzed by the method of standard additions, with standard manganese added directly in the furnace. Appropriate additions for serum were found to be 50 and 100 pg, provided as 10 p1 of the 5 ng/ml and 10 ng/ml standards, respectively. CSF was similarly analyzed, using 25 and 50 pg additions,provided as 5 p and 10 p1 of the 5 ng/ml standard,respectively.Results presented are total manganese minus added manganese.
RESULTS AND DISCUSSION The parameters shown in Table I were selected after extensive testing and were verified as follows. Argon Flow Setting. The absorption signal produced by manganese in the furnace is sensitive to the rate of purge gas flow, with signals more than doubling as the flow rate is decreased from 80 to 18 cm3/min. Low flow rates markedly 1470
0.64, 0.64, 0.66, 0.70, 0.78, 0.89
800
Temperature ('C)
shorten graphite cylinder lifespan, however. T h e flow rate selected allowed approximately 100 runs with acceptable peak height per cylinder. Although further signal increases could be obtained by completely interrupting the flow a t the time of atomization, the resulting signal peaks were irregularly shaped and poorly reproducible, probably a consequence of erratic background correction in such a flow mode. Therefore, the constant flow mode was selected. Slit Width. Operation with the indicated value permitted the inclusion of the absorbing manganese triplet (279.5 nm, 279.8 nm, 280.1 nm) and provided increased deuterium arc intensity without detectable changes in signal linearity or the introduction of spurious signals. D r y i n g P a r a m e t e r s . For serum and CSF, the drying conditions indicated were found t o produce rapid drying without spattering. Serum required more vigorous drying t o prevent spattering a t the start of the charring stage. C h a r r i n g P a r a m e t e r s . Proper conditions for the charring stage are of extreme importance in flameless determinations. Optimization requires temperature and time sufficient t o volatilize nonspecific background constituents inherent in the various fluid matrices without volatilizing the element of interest. Subsequent deuterium arc background correction may be required for full compensation. Figure 1 depicts the effect of charring parameters on the signal produced by 500 pg of manganese (as 10 p1 of 50 ng/ml standard). Clearly, manganese in the standard (aqueous)
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
Table IV. Reported Normal Manganese Concentrations in Serum and CSF' Sample
Method
Manganese (nglml)
Source
Serum 1.02 (0.74-1.25) Flameless AAS Serum 13.4 (6.5-18.0) Flameless AAS Flameless AAS Serum 1.94 (median = 1.58) Serum 11.0 Flameless AAS Serum 21.8 (8.5-40.5) Flameless AAS Serum 24.0 (12.0-38.0) FlameAAS * Serum 9.7 (S.D. = 4.0) Neutron activation Serum 0.587 (S.D. = 0.183) Neutron activation Serum 13.0b (11-14) Neutron activation Serum 0.57 (0.38-1.04) Neutron activation Serum 0.63 (0.36-0.90) Neutron activation CSF 0.66 (0.55-0.89) Flameless AAS CSF 1.16 (0.83-1.50) Neutron activation a Values given are the means taken directly or calculated from the published data. tion.
matrix is not appreciably volatilized until the 60 s/800 "C charring is exceeded. Consequently, even before other limiting factors are examined, the 60 s/800 "C setting is the maximum allowable limit if these standards are t o be used. Next, it is useful to observe the background signal produced by various volumes of serum or CSF as a function of charring parameters. This background was determined by operating with a lead lamp a t the nearly 280.2 nonabsorbing wavelength, and the results are shown in Figure 2. It is clear that a 50-pl sample of these biological fluids gives rise t o a large background signal which cannot be completely eradicated by charring alone, even a t the 60 s/800 "C setting, and additional background correction with the deuterium arc must be employed. Complete background correction points ( A < 0.001) were determined by measuring background as before a t the lead wavelength, but with the additional operation of the deuterium arc, and these are given in Table 11. Finally, the manganese lamp was inserted and the wavelength reset t o the absorbing manganese triplet. Possible loss of manganese due to volatilization during the charring stage was investigated in both sample matrices. Samples of serum and CSF were analyzed for true manganese a t successively increasing charring parameters, beginning with the respective minimum correction points. T h e results are shown in Figure 3, and it is apparent t h a t the metal is not appreciably volatilized a t or below the 60 s/800 "C setting for either matrix. Although any of the charring parameters in this range are equally acceptable, the 60 s/800 "C setting was chosen for maximum reduction of background signal prior to deuterium arc correction. Atomization P a r a m e t e r s , Although a wide range (2100-2700 "C) of atomization temperatures can be employed, the 2300 "C setting chosen was found to produce acceptable peak height and reasonable cylinder lifetime. T h e 8-s time period was long enough to allow complete return of signal to baseline values and yielded subsequent blank runs ( A
