Apr 7, 1988 - Fluonmetry of Selenium in Body Fluids after Digestion with Nitric Acid, Magnesium ... Filter the aqueous (lower) phase into a second funnel.
2. McFarland
KF, Catalano
EW, Day JF, Thorpe
Nonenzymatic glucosylation of serum proteins Diabetes 1979;28:1011-4. 3. Yue DK, Morris K, McLennan S, Turtle
SR. Baynes J.
in diabetes
JR Glycosylation of plasma protein and its relation to glycosylated hemoglobin in diabetes. Diabetes 1980;29:296-300. 4. Fl#{252}ckiger R, Winterhalter KH. In vitro synthesis of hemoglobin A1c. FEBS Lett 1976;71:356-60. 5. Johnson RN, Metcalf PA, Baker JR. Fructheamine: a new approach to the estimation of serum glycosylprotein. An index of diabetic control. Clin Chim Acta 1982;127:87-95. 6. Rendell M, Kao G, Mecherikunnel P, et al. Use of aminophenylboronic acid affinity chromatography to measure glycosylated albumin levels. J Lab Clin Med 1985;105:63-9.
CLIN. CHEM. 34/9,
1908-1910
7. Vogt BW, Schleicher glucose in human 1982;31:1123-7.
mellitus.
ED, Wieland OH. a-Amino-lysine-bound obtained at autopsy. Diabetes
tissues
8. Schleicher E, Wieland OH. Specific quantitation by HPLC of protein (lysine) bound glucose in human serum albumin and other glycosylated proteins. J Cliii Chem Clin Biochem 1981;19:81-7. 9. Finot PA, Mauron J. Le blocage de la lysine par Ia reaction de Maillard. I. Synthese de N-(desoxy-1-D-fructosyl-1)et N-(desoxy-1D-lactulosyl-1)-L-lysines. Helv Chin Acts 1969;52:1488-95. 10. Finot PA, Bricout J, Viani R, Mauron J. Identificationof a new lysine derivative obtained upon acid hydrolysis of heated milk. Experientia 1968;24:1097-9. 11. Hodge JE. Amadori rearrangement [Review]. Adv Carbohydr Chem 1955;10:169-205.
(1988)
Fluonmetry of Selenium in Body Fluids after Digestion with Nitric Acid, Magnesium Hexahydrate, and Hydrochlonc Acid Jean Petterason,
Lena Hanseon,
Ult Ornemaric,
and Ak. Olin
A digestion procedure involving nitric acid, magnesium nitrate hexahydrate, and hydrochloric acid suffices for seleniurn determinations in whole blood, serum, and urine by molecular
fluorescence
spectrometry.
To test the accuracy
of
the method we compared the results with those from hydridegeneration atomic absorption spectrometry, and we also analyzed reference materials. Additional Keyphraaes: trace elements cence spectromet,y hydride-generation tromefty compared urine ‘
molecular
atomic
fluores-
absorption spec-
About half determinations lar fluorescence
of those laboratories involved in selenium in blood perform their analyses by molecuspectrometry (MFS) (1,2). The procedure is usually based on the reaction between tetravalent selenium and 2,3-diaminonaphthalene at a pH between 1 and 2. A fluorescent complex, 4,5-benzopiazaelenol, is formed, which is extracted into an organic phase (usually cyclohexane), where its fluorescence is measured. Most often, the sample digestion is performed with an acid mixture including perchloric acid (e.g., 3). Efforts to replace this hazardous but effective agent for destruction of biological material include trials of a combination of phosphoric acid, nitric acid, and hydrogen peroxide (4). A digestion procedure based on a mixture of magnesium nitrate, nitric acid, and hydrochloric acid for the determination of selenium in blood and urine samples has proved suitable when combined with hydridegeneration atomic absorption spectrometry (HG-AAS) (5). Here we report the compatibility of this digestion procedure with the well-established MFS method for determining selenium.
Department of Analytical Chemistry, University of Uppeala, Box 531, 8-751 21 Uppsala, Sweden. Received April 7, 1988; accepted May 24, 1988. 1908
CLINICAL CHEMISTRY,
Nitrate
Vol. 34, No. 9, 1988
P.O.
MaterIals
and
Methods
Apparatus We used the HG-AAS instrumentation described previously (6), a fluorescence spectrometer (Perkin-Elmer Model 204) equipped with a xenon lamp, and 1.0-cm (pathlength) quartz cuvettes for all measurements. A temperature-regulated aluminum block (6) was used for digestion, reduction, and reaction. Reagents
and Materials
All acids and reagents were of”pro analysi” quality except for the diaminonaphthalene (Fluka Chemie AG, Buchs, Switzerland), which had to be purified just before use as follows. To 50 mL ofde-aerated 0.1 mol/L HC1 add 50 mg of diaminonaphthalene and 250 mg of hydroxylammonium chloride. Heat this mixture at 50 ‘C for 15 miii, cool, and ifiter the solution into a separatory funnel. Add 10 mL of cyclohexane, shake for 1 mm, and let the phases separate for 5 mm. Filter the aqueous (lower) phase into a second funnel containing 10 mL of cyclohexane and repeat the extraction. This time, ifiter the aqueous phase into a 50-mL screwcapped test tube. Bubble nitrogen gas through the mixture for 2 miii, and cap the tube.. The indicator solution was prepared by dissolving 0.3 g of gentian violet in 100 mL of water; the 36 mmolJL EDTA solution was prepared by dissolving 6.7 g of Na2EDTA H2O in 500 mL of water. Selenium standard solutions were prepared as described in ref. 6. The reference materials were Standard Reference Material no. 1577a (bovine liver; U.S. National Bureau of Standards, Gaithersburg, MD) and Seronorm 105 (human serum) and 108 (human urine) from Nycomed AS, Oslo, Norway. The ifiter paper used was of medium pore size (no. OOM; Munktell, Grycksbo, Sweden).
Procedure Digestion. Weigh the sample (about 0. 1 g of lyophilized blood, 1 g of serum, or 1 g of urine) into a 50-mL screwcapped test tube. Add 4 g of Mg(N03)2 6H20, 10 mL of concentrated nitric acid, and 1 mL of 6 molIL hydrochloric acid. Heat in an aluminum block in the following sequences of time (h) and temperature (#{176}C): 1-50, 1-85, 1-105, 1-125, 2-175, 2-225, 2-300, and 3-500. Then cool the mixture to room temperature. Reduction. Add 10 mL of 6 mol/L hydrochloric acid. Cap the test tubes and heat for 1 h at 130 #{176}C. pH-adjustment. Adjust the hydrogen ion concentration to about 30 mmol/L by first adding 3.5 mL of6 molIL ammonia reagent, then 0.5 mL of the indicator, 1 mL of 36 mmol/L EDTA, and finally ammonia reagent (2 molIL solution) until a green color is visible. Reaction and extmction. Bubble nitrogen gas through the digested and reduced sample for 30 s, then add 1 mL of diaminonaphthalene reagent and 5 mL ofcyclohexane. Cap the test tube and place it for 30 miii in the aluminum block, which has been preheated to 60 #{176}C. Cool to room temperatore, shake for 1 mm, and transfer the organic layer to a test tube after 5 mm. Measure the fluorescence of the sample (excitation wavelength 375 nm, emission wavelength 522
Table 1. Blank Signals Successive Purifications
in the MFS Procedure after of the Diaminonaphthaiene Reagent Blank signal, arb. fluor. units
.
The must
method
n 16
Mean 26
SD 14
14
11
2
23
3
1
820 g of selenium per liter yields about 100 arb. fluorescence “With
Table
units.
cyclohexane.
2. SelenIum Whole Blood
Concentration (pg/g) and Three Reference
HG-AAS
MFS
Sample Lyophilizedwhole blood Seronorni 1058 Seronorm lO8’ SAM no. 1577ac
n
8 4 2 2
Mean 0.568 0.085 0.048 0.678
in Lyophlllzed Materials
SD
0.026 0.004 0.002 0.007
Mean 0.564 0.088 0.048 0.695
SD 0.015
0.002 0.001 0.002
Human serum, certified 90 ± 6 M9/L. “Human urine, certified 49 j9/L. C Bovine liver, certified 0.71 ± 0.07 zg/g.
ma).
Results
Purification
Three extractions” Three extractions, and reagent dissolved in de-aerated HCI Three extractions, and reagent dissolved in de-aerated HCI and fiftered
and
DIscussIon
reduction be sure
to selenite. After digestion, one all the selenium in the sample is in the This is the only form of selenium that
of selenate that
tetravalent state. reacts with diaminonaphthalene to give the desired fluorescent species. The reduction is accomplished by reacting the digested sample with hydrochloric acid. Ifthe reaction tubes are open during the reduction, some ofthe hydrochloric acid added will boil off. This is not a problem in the HG-AAS procedure, but in the MFS method it will cause inconvemence. The temperature varies somewhat with the positions in the aluminum block, and different amounts of hydrochloric acid will leave each test tube. This makes the pH-adjustment in the MFS procedure more time consuming, and the reduction should therefore preferably be performed in closed vessels. The blank. The dianunonaphthalene reagent is unstable, and its rate of decomposition increases with temperature, exposure to daylight, and presence of oxidizing agents. High and h-reproducible blank signals result unless an efficient purification procedure is applied and the purified product is stored under proper conditions. A fluorescing polymer of diaminonaphthalene is held responsible for the undesired blank signals (7). Several purification procedures have been described in the literature (e.g., 8,9). We found it necessary to purify the diaminonaphthalene reagent by two repeated extractions with cyclohexane, to filter the solution after each extraction, and, last but not least, to deoxygenate the hydrochloric acid used to dissolve the diaminonaphthalene by bubbling with nitrogen gas to ensure low and reproducible blank signals. Hydroxylammonium chloride was also added to the reagent as an antioxidizung agent. Table 1 shows the improvement with the successive purification steps. The pH adjustment. A pH adjustment is necessary before the complexation reaction between selenite and diamunonaphthalene. A pH of about 1.8 is generally considered optimal (see e.g., 7). The very high salt concentrations
method
after makes
digestion and reduction in the proposed proper pH measurement difficult. Therefore, acidity should rather be expressed as a hydrogen ion concentration than as a pH value. For convenience, however, we use the pH notation understood to mean -log[H+J. We found that a pH value between 1 and 2 for the reaction only slightly
influenced
obtained
the fluorescence
intensity.
Outside
these
iimits the fluorescence declined considerably. The pH adjustment described in the experimental section gives a final pH value of 1.5 ± 0.2, which is sufficiently accurate. It is important that the pH be promptly adjusted after the indicator has been added, because its color begins to fade after about an hour. Effect of reaction time and temperature. Optimum conditions for the complexation reaction between diaminonaphthalene and selenite were found to be 30 mm at 60 #{176}C. Calibration. The calibration curve was established from known amounts ofselenite subjected to the whole procedure, from digestion to fluorimetry. It is, however, not necessary to include the digestion step if all reagents are added to the calibration solutions and the pH is adjusted to the value used for the samples in the complexation step. Analytical results. Table 2 gives results from the analysis of lyophilized whole-blood samples by fluorimetry and hydride-generation atomic absorption spectrometry. Both MFS and HG-AAS were applied to each digest and all digestions were performed simultaneously. Table 2 also includes the results obtained from analysis of the three reference materials. Results from the two methods overlap, and results for the reference materials were satisfactory. We conclude that the described digestion procedure is a valid alternative to more common destruction methods (often including perchioric acid) when selenium is to be determined in biological material by fluorimetry. CLINICAL
CHEMISTRY,
Vol. 34, No. 9, 1988
1909
References 1. Ihnat M, Wolynet.z MS, Thomassen Y, Verlinden M. Interlaboratrial on the determination of total selenium in lyophilized human blood serum. Pure Appi Chem 1986;58:1063-76. tory
2. Koh T-S. Effects of blood standards on interlaboratory variation in the assay of blood selenium. Anal Chem 1987;59:1597-9. 3. Tamari Y, Ohmori S, Hiraki K. Fluorometry of nanogram amounts of 1986;32:1464-7.
selenium
in
biological
samples.
Clin
Chem
4. Reamer DC, Veillon C. Elimination of perchloric acid in digestion of biological fluids for fluorometric determination of selenium. Anal Chem 1983;55:1605-6. 5. Hansson L, Pettersson J, Olin A. A comparison of two digestion
CLIN.
CHEM.
34/9, 1910-1912
W. Ryder,’
and Shaking
Stephen J. Jay,2 MeMn
and increased results for pH for both manufacturers’ ampules with the highest CO2 tensions, and this bias was not offset by increasing the shaking rate. We conclude that both storage temperature and shaking rate must be precisely defined and carefully monitored before’ these products are used in a quality-control program. Commercially available aqueous pH and blood-gas control products frequently are used in quality-control programs. However, the pre-analytical variables associated with handling and transferring these products to the analytical device are considered more likely to produce significant errors than are the variables intrinsic to the measurement itself (1). Unfortunately, the manufacturers of two of these products, “contrlL” and “G.A.S.” blood-gas controls, give somewhat ambiguous directions regarding proper handling of the ampules before analysis. Instructions call for these materials to be stored at “room temperature” (2) and for the operator to “shake” (3) or “vigorously shake” (2) them before analysis. We noted a considerable difference among operators as to what constitutes “vigorous” shaking, and a review of temperature records showed that “room temperature” in ‘Department of Pathology, Indiana University School of Medicine, Wishard Memorial Hospital, 1001 West 10th Street, Indianapohs, iN 46202. 2Division of Academic Affairs, Methodist Hospital, Indianapolis, 18, 1988; accepted
CLINICAL CHEMISTRY,
Acta 1982;134:417-9.
Results for Two
R. GiIck,1 and John R. Woods2
kO2
1910
Anal Chin
Rate on pH and Blood-Gas
Directions for pre-analytical handling of ampules of two commercially available aqueous quality-control products (contrlL and G.A.S.) contain vague instructions such as “store at room temperature” and “shake vigorously” before analysis. We examined the effect of different storage ternperatures (25, 31 , and 38 #{176}C) and shaking rate (one, two, and four shakes per second) on pH and blood-gas results. For both products, increasing the storage temperature significantly decreased p2 results, the magnitude of the bias being greatest for those solutions with the highest 02 tensions. However, increasing the shaking rate partly offset this bias. Increasing storage temperature also decreased results for
IN. Received February
selenium.
(1988)
Effect of Storage Temperature Quality-Control Products Kenneth
procedures for the determination of selenium in biological material. Talanta 1987;34:829-33. 6. Pettersson J, Hansson L, Olin A. Comparison of four digestion methods for the determination of selenium in bovine liver by hydride generation and atomic-absorption spectrometry in a flow system. Talanta 1986;33:249-54. 7. Bayfield RF, Romalis LF. pH control in the fluorometric assay for selenium with 2,3-diaminonaphthalene. Anal Biochem 1985;144:569-76. 8. Tamari Y. Fluorimetric determination of nanogram amounts of selenium in rocks. Bunseki Kakagu 1984;33:E115-22. (In English.) 9. Watkinson JH. Improvements to the sensitivity, resolution and blank value in the semi-automatic fluor#{252}netricdetermination of
May 26, 1988.
Vol. 34, No. 9, 1988
our laboratory was quite variable. We thus examined the effect of these two pro-analytical variables on pH and bloodgas results. In previous reports, these variables received little attention. Komjathy et al. (4) and Abramson et al. (5) advised shaking ampules “vigorously” for lOs before analysis; Leary et al. (6) reported shaking them for 30 s. In no case was “vigorous” defined, nor were any data provided to support these statements. Mans et al. (7) advised keeping the ampules on the bench for a day before use and at 25 #{176}C for 30 miii before analysis. Komjathy et al. (4) stored ampules at “ambient room temperature for at least 12 h before testing.” Leary et al. (6) compared pH and blood-gas results when ampules were incubated at 22 and 30#{176}C for 5 mm before analysis and found that, at higher gas tensions, the measured po, decreased with increasing temperature. The potential interaction of these two pre-analytical variables, shaking rate and storage temperature, has not been examined. Here we report the effect of storage temperature and shaking frequency on pH and blood-gas results for aqueous quality-control materials from two different manufacturers. Materials
and
Methods
Materials We purchased the blood-gas and pH quality-control materials from their manufacturers: General Diagnostics Blood G.A.S. (Gas Analyzer System) control from Organon Teknika Corp., Morris Plains, NJ 07950, and contriL Blood Gas Control from Instrumentation Laboratory, Lexington, MA 02 173. Each product consists of a set of three ampules, each ampule having different pH values and different oxygen and carbon dioxide tensions. However, the G.A.S. ampules with the lowest pH (acidosis) have the highest oxygen and lowest carbon dioxide tensions, whereas the contrlL controls with the highest oxygen and lowest carbon dioxide tensions have the highest pH (alkalosis). instrumentation We Model
made all pH and blood-gas measurements 813 Blood Gas Analyzer (Instrumentation
with a Labora-
