Anal. Chem. 1982, 54,595-596
Table I. Comparison of the Mean Mirex Concentra. tions in Lake Trout and Lamprey by Four Different Techniquesa trout lamprey rereported mirex ported mirex mean,“ concn,b mean: concn,b technique PgIwL wglg PgIwL wglg EC-GC 12!3 0.65 17.2 0.086 MS-SIM 1113 0.03 17.2 0.004 m/z 546 MS-SIM 12’7 0.64 16.5 0.083 m/z 272 MS-SIM 126 0.63 14.4 0.0’72 4 ions ( m / z 203, 237, 238, 272) a The mean mirex concentrations are reported for 10 trout and 5 larnprey samples in Table I1 of Onuska et al. ( 1 ). Converted from the reported mean for trout and lamprey in Table I1 of Onuska et al. (1 ) using the sample sizes, final volumes, aliquot volumes, and injected volumes described in the Experimental Section.
!595
which we present in Table I clearly illustrate the fact that the EC-GC technique, and the MS-SIM at m / z 272 and at four ions (mlz 203, 237,238, and 272) techniques are not suitable substitutes for the MS-SIM at m / z 546 technique for the determination of mirex in biological samples from the environment. The determinatioln of mirex in fish and other environmeiital samples from heavily contaminated water cannot be talken lightly and must be supported by confirmatory mass spectral evidence. Under ordinary conditions, the acquisition of adequate full mass spectral data to unequivocally establish the presence of mirex is difficult. Even the interpretation of diata for its quantification can be complicated. Therefore, judicious scrutiny of all confirmatory and quantitative data for mirex is essential in order to minimize the release of reports which are in error. LITERATURE CITED Onuska. F. I.: Comba, M. E.; Coburn, J. A. Anal. Chem. 1980, 52, 2272-2275. Laseter, J. L.: DeLeon, I. R.; Remele, P. C. Anal. Chem. 1978, 5 0 , 1. 169-1 . - - . 172. . . -. DeLeon, I. R.; Warren, V.: Laseter, J. L. Ouant. Mass Specfrom. Life Sci. 1978, 2, 483-492. Kaiser, K. L. E. Sclence 1974, 186, 523. Norstrom, R. J.: Nallet, D. J.; Onuska, F. I.; Comba, M. E. Environ. Scl. Techno/. 1980, 14, 860-886.
authors evade the issue of providing conclusive mass spectral data for mirex by stating that the mass spectra of mirex and its degradation products are presented elsewhere (5). However, the mass spectral data for mirex is not presented as alleged (5). The issue is further complicated by the presentation of,selected-ion-detection traces for the m / z 203, 237, 238, and 272 ions, whereas the selected-ion-detection traces for the m/z 546 are selectively omitted. On the basis of our experience with mirex analysis (2, 3) and the data presented, it is our opinion that Onuska et al. have not successfully shown that the method described is appropriate far mirex determinations. The corrected data
Center for Bio-Organic Studies University of New Orleans Lakefront New Orleans, Louisiana 70148
Sir: With regards to our paper “Quantitative Determination of Mirex and Its Degradation Products by High Resolution Capillary Gas Chromatograph/Mass Spectrometry” ( I ) , we wish to express our views on the comments by J. L. Laseter and I. R. DeLeon. The first point of concern is their calculation of mirex concentrations on a p g / g basis. With reference to the calculations performed by Laseter on the data in our Table 11, we hope to provide some clarification. The choice of pg/pL as an expression of concentration was used to avoid any specific implication or reference to mirex levels in Lake Ontario biota, since this was not the intent of the article. In hindsight this may have been unfortunate, since, as in Laseter’s case, the data may be misinterpreted. However it should be noted that none of the original reviewers, including Laseter, questioned this presentation. The data on extrapolated values (pg/g) presented in Table I by Laseter et al. are incorrect for the m / z 546 technique. He has taken data which have already been normalized to enable comparison with the other three techniques and reapplied the volume coirrection factor of 20 (1 mL to 50 pL) to give a result of 0.03, which should read 0.60 pg/g. The data presented in our Table I1 on the mirex levels in lamprey samples were also normalized. Analyses again were performed on concentrated portions for the m/z 546 SIM
analysis and the four ion SIM analysis and on unconcentrated portions for the other two techniques as indicated by the footnotes in Table 11. It is incorrect to reapply this volume factor in calculating the corresponding concentrations expressed on a pg/g Ibasis. The procedures used in this paper do not reflect a 20-fold difference in resultla implied by Laseter and DeLeon and we have not experienced the interferences on the m / z 546 ion mentioned by them. Our data on m / z 546 revealed none of these interferences, perhaps due to superior cleanup and fractionation techniques used in our laboratory. Our other concern deals with the issue of “conclusive tividence for the presence of mirex”. We feel that the data and the mass spectrometry ideintification is conclusive for mirex. Millard (2) estimates a probability greater than loe for single ion determinations on mlz values higher than m/z 200, with one retention time. Our MS data indicated that the ion ratios were the same for the environmental samples as for our reference standard, since quantitation on all ions produced the same results. The correct ratios for the four selected ions and an ion from the molecular cluster present to us conclusive evidence for the presence and level of mirex reported in our paper. The additional confirmation of retention indices on four different chromatographic columns strengthens our position.
0003-2700/82/0354-0595$01.25/0
John L. Laseter* Ildefonso R. DeLeon
RECEIVED for review March 23, 1981. Resubmitted October 19, 1981. Accepted October 19, 1981.
0 1982 American Chemlcel Soclety
596
Anal. Chem. 1982, 5 4 , 596-598 (2) Millard, B. J. "Quantitative Mass Spectrometry"; Heyden: London, 1978.
It was our judgement that the inclusion of a spectrum of mirex and the trace of the m / z 546 response were not required to substantiate these results. This explanation appears to have satisfied the majority of reviewers, since the article was publisKed as such after our written comments. In conclusion, we feel that our regression analysis data indicate that the MS-SIM and GC-EC methods of operation can provide reliable results, using the described procedure.
F. I. Onuska* M. E. Comba J. A. Coburn National Water Research Institute Canada Centre for Inland Waters and Inland Waters Directorate 867 Lakeshore Road Burlington, Ontario, Canada L7R 4A6
LITERATURE CITED
RECEIVEDfor review October 26, 1981. Accepted November 24, 1981.
(1) Onuska, F. I.; Comba, M. E.: Coburn, J. L. Anal. Chem. W80, 52, 2272-2275.
AIDS FOR ANALYTICAL CHEMISTS Optically Discriminating Flow-Through Cuvette Wolfgang Vogt, * Slegmund Lorenz Braun, Siegfried Wilhelm, and Helmut Schwab Institut fur Klinische Chemie, Klinikum Grosshadern, Marchioninistrasse 15, 0-8000Munchen 70, Federal Republic of Germany
The introduction of continuous flow analyzers by Skeggs in 1957 ( I ) brought practical automation to the laboratory for the first time. A necessary condition in this technique is the segmentation of the fluid stream by air bubbles to avoid mixing of the successive samples. These air bubbles make the photometric measurement of the segmentated fluid stream more difficult. T o overcome this problem several kinds of cuvettes have been developed. The first one was a device in which the air is removed previous to entering the flow cell by venting with a simple T-shaped glass fitting (2). This debubbling system has some disadvantages: First, the debubbling causes long half-wash time and thus interaction between samples increases. Second, the debubbler waste line carries always an aliquot of the fluid. Third, air bubbles are occasionally pulled through the flow cell which can lead to wrong measurement. Therefore it was desirable to eliminate the debubbler step before measurement in order to gain more rapid attainment of steady-state conditions, this means lower interaction between samples and maintenance of the integrity of the segmented analysis stream, permitting subsequent handling and remeasurement. Habig and co-workers developed a conductance-measuring device for detecting an air bubble when it is in the flow cell (3). They demonstrated the dramatic improvement in wash obtained when air bubbles are passed through the flow cell. Technicon has incorporated into the AutoAnalyzers of the third generation, the SMAC system, a bubble-through flow cell with a very small volume of only 2 pL (4). The debubbling function for the output record is performed by a software recognition of the large absorbance changes created by a bubble. Although this change in absorbance is a large one, it is not always clearly defined. Therefore we developed a flow cell with optically discriminating properties, which will provide total reflection of the light beam when air fills the flow cell (5). EXPERIMENTAL SECTION The flow cell consists in its optically effective parts of two parallel quartz prisms with a 3.2 mm broad space between them. The two prisms, the upper and lower cover are fused together with glass-powder forming a 13 mm long space between them (Mikro-Glastechnik, Hartheim, G.F.R.). The fluid stream is 0003-2700/82/0354-0598$0 1.25/0
pumped through this space (Figure 1). The height of the flow cell is 1.5 mm. Its total volume is 60 pL. The inlet and outlet holes are drilled in the upper cover of the flow cell. The inlet hole is 1.6 mm in diameter whereas the outlet has 3.2 mm in order to facilitate venting of the air bubbles. The inner surfaces of the flow cell have to be very smooth to obtain quick passing and to avoid sticking of the air bubbles. The upper and the lower cover of the flow cell are made of black quartz to minimize reflections. The flow cell mounted on a holder with diaphragms at each end is placed into a common filter photometer from Labotron (Geretsried, G.F.R.). The measured absorbances are fed into an absorbance discriminator, characterized by a sample and hold circuit. Every air bubble produces a very sharp increase in absorbance. These signals will easily be recognized and neglected by the absorbance discriminator. Thus only the interesting absorbances of the fluid filled segments are recorded. RESULTS AND DISCUSSION Principle and Function. The light beam passes through the fluid-filled flow cell with a small refraction of 5O for red light and 8' for blue light (Figure 2). The light path is about 5 mm in length. Thirty microliters is the minimal fluid volume and therefore the optical effective volume to obtain correct absorbance values and to avoid uncontrolled refractions of the light beam on the surface between fluid and air bubble. In an air-filled flow cell we have total reflection of the light beam because of the prismatic construction of the flow cell (Figure 3). The limiting angle of total reflection between quartz and air is 4 3 O . The limiting angle between quartz and water is 66O. Thus there is a broad security range for fixing the cuvette correctly in a holder. In order to reach this angle of total reflection when air bubbles are passing through the flow cell, we have chosen an angle of incidence of 50' for the light beam. Therefore, in the condition of the air-filled flow cell, the light beam must be totally reflected and no light reaches the photomultiplier. Thus the absorbance signal from the photometer reaches values close to infinity. In contrast to other cuvette systems, we have a clearly defined signal, when air bubbles fill the flow cell, as needed in the continuous flow analyzers commonly used. The optically effective pathlength of the flow cell depends on the refraction index of the solution to be measured. Since 0 1982 American Chemical Society
