CLINICAL TOXICOLOGY 11(2), pp. 159-171 (1977)
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A Completely Automated Fluorometric Blood Cyanide Method: A Specific Assay Incorporating Dialysis and Distillation*
WILLIAM A. GROFF,? SAMUEL A. CUCINELL, M. D. ,$ PASQUALE VICARIO, and ANDRIS KAMINSKIS Biomedical Laboratory Edgewood Arsenal Aberdeen Proving Ground, Maryland
*The views of the authors do not purport to reflect the position of the Department of Defense. The use of trade names in this r e p o r t does not constitute an official endorsement o r approval of the u s e of s u c h c o m m e r c i a l hardware o r software. This report may not be cited f o r purposes of advertisement. The volunteers who donated blood for these t e s t s a r e enlisted U.S. Army personnel. These t e s t s are governed by the principles, policies, and rules f o r medical volunteers as established in AR 70-25, and the Declaration of Helsinki. In conducting the r e s e a r c h described in this report, the investigat o r s adhered to the "Guide f o r the C a r e and Use of Laboratory Animals" as promulgated by the Committee on Revision of the Guide f o r Laboratory Animals Facilities and C a r e of the Institute of Laboratory Animal Resources, National Research Council. ?Reprint requests should be a d d r e s s e d to William A. Groff, Biomedical Laboratory, Edgewood Arsenal, APG, Maryland 2 1010. $Present address: Box 0, Gorgas Hospital, Panama Canal Zone. 159 Copyright 0 1977 hy MarLel Dekker. Inc All Rights Reserved Neither this work nor any part may he reproduced or transmitted in any form or by any means. electronic or mechanical. including photocopying, microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher
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160
GROFF E T AL.
The automated blood cyanide method described in this report was developed to meet the requirements of o u r laboratory f o r studies of cyanide poisoning and its therapy. Since existing automated CN- methods [ 1-41 are designed for the a ss ay of water samples, an extensive literature survey was made in o rd er to select the most sensitive and specific method that could be adapted to a practical, automated as s ay of blood CN-. The Prussian blue [5-71, picric acid [8], and copper-benzidine [9] reactions are not sufficiently sensitive to meet our requirements. The o-cresolphthalein [ 101 and phenolphthalein [ 111 methods lack specificity. The methods of Aldridge [12-141 and Epstein [15, 161, based on the Konig [17] reaction, a r e widely used and p o s s e s s adequate sensitivity, but utilize reagents which rapidly deteriorate Tygon plastic tubing. Two fluorometric procedures, one by Hanker, Gamson, and Klapper [ 181 and the other by Hanker, Gelberg, and Witten [ 191, appeared desirable, due to extreme sensitivity and mild reagents. The automation of the latter, with the addition of dialysis and distillation s tep s to eliminate interfering substances, is the subject of this report. M A T E R I A L AND M E T H O D S Reagents 1. Sodium hydroxide, 0.03 N. Dissolve 1.2 gm sodium hydroxide in distilled water. Add 2.0 m l of B r i j 35 and dilute to 1 l i t e r . 2. Sodium hydroxide, 0.5 N. Dissolve 20.0 gm sodium hydroxide in distilled water and dilute to 1 liter. 3. Glycine buffer. Dissolve 77.4 gm glycine and 58.6 gm sodium chloride in distilled water. Add 1.0 ml of Brij 35 and dilute to 1 liter. 4. Palladium chelate, 0.01 gm/liter. Dissolve 0.010 gm potassium b is ( 5-sulfoxino)palladium (11)* in distilled water. Add 1.0 ml B r i j 35 and dilute to 1 liter. The chelate was prepared as follows [ 191 : 8-hydroxy- 5-quinoline sulfonic acid (4.50 gm, 0.02 mole) was added to a solution of palladous chloride (2.14 gm, 0.01 mole) in 300 ml of 5% sulfuric acid. The solution was heated to boiling and then cooled to room temperature. Saturated potassium carbonate solution was added until the evolution of carbon dioxide ceased. The chelate separated as a fine, yellow p r e cipitate. It was collected on a filter, washed successively with 10% potassium carbonate solution, water, alcohol, and ether; then dried in air. *Prepared as described by Hanker et al. [ 191.
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5. Magnesium chloride, 1.0 gm/liter. Dissolve 1.0 gm magnesium chloride in distilled water. Add 1.0 Brij 35 and dilute to 1 liter. 6. Propanol, 50%. Dilute 500 ml propanol to 1 l i t e r with distilled water. 7. Acetic acid, 1.0 N. Add 60.0 ml concentrated acetic acid to distilled water. Add 2.0 ml Brij 35 and dilute to 1 liter. 8. Heparin solution, approximately 90 units/ml. Add 10.0 ml heparin (1000 units/ml) to 100 ml s t eri l e isotonic saline. 9. Brij 35. Surfactant obtained from Technicon Instruments Corporation, Tarrytown, New York. 10. Stock potassium cyanide solution, 38.4 mmole/liter. Dissolve 0.2 503 gm potassium cyanide in 0.1 N sodium hydroxide and dilute to 100 nil with 0 . 1 N sodium hydroxide. This solution contains 1 mg of CN- per milliliter. Crystalline potassium cyanide is deliquescent and should be stored under d r y conditions. APPARATUS The analytical system consists of the following modules from Technicon Instruments Corporation, Tarrytown, New York: ( a ) s ampler 11, ( b ) model I1 proportioning pump, ( c ) dialyzer (37 "C) with one s e t of dialyzing plates and type C membrane, ( d ) heating bath ( 9 5 ' C ) with a 40-ft glass coil, ( e ) one 20-ft time-delay coil, ( f ) fluorometer In with Corning Glass filters (excitation - #5970 and emission - #4308 and #3389), and ( g ) reco rd e r with percent transmittance paper. PROCEDURE The flow diagram of the automated manifold is shown in Fig. 1 as it is used for intermittent sampling of blood collected by syringe o r Vacutainer. * Sample and water wash cups are placed alternately on the sample platter, which is operated at a ra t e of 40/hr, resulting in a n effective sampling rate of 20/hr. For continuous in vivo monitoring, the Sampler I1 module and intermittent sample pump tube are disconnected. A 0.235 ml/min heparin pump tube and a 0.60 ml/min in vivo sample pump tube a r e attached to the in vivo sampler [20] shown in Fig. 2, using 4-ft lengths of 0.015-in. i.d. Tygon tubing (Technicon stock #116-0536-04). The
*Available from Scientific Products, 8855 McGaw Rd., Columbia, Md. 21045.
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RECORDER
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Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
164
GROFF E T AL.
in vivo sample pump tube, which draws a mixture of blood and heparin, is attached to the H3 cactus in place of the intermittent sample pump tube. The blood sample is mixed f i r s t with sodium hydroxide and the erythrocytes completely hemolyzed in a l arge mixing coil before entering the dialyzer. In alkaline solution, CN- is released both from cyanmethemoglobin and from nonspecific binding. The hemolyzate is then dialyzed a t pH 12 and a constant fraction of the CNpresent in the sample s t ream p as s es into the recipient s t r e a m which exits from the dialyzer. The recipient s t r e a m is acidified before entering the 95°C heating bath. During this 50-sec distillation period at pH 4, a constant fraction of the CN- is volatilized into the air bubbles that segment the liquid. This vapor must not come in contact with Tygon plastic tubing, due to reaction and l o s s of the CN-. Therefore, the portion of the manifold from point A to point B on the flow diagram must be constructed of glass-to-glass fittings and connections. The vapor and liquid phases are separated by means of a C2 connector following the addition of propanol to eliminate foaming. A portion of the vapor phase, which is withdrawn from the C2 connector by a difference in flow rates, is used to segment a stream of sodium hydroxide. After the absorption of CN- in alkali, plastic tubing may again be used without l o s s of CN-. An aliquot of the alkali, which contains absorbed CN-, is mixed f i r s t with buffer and then with palladium chelate reagent. CN- demasks the nonfluorescent potassium bis ( 5-sulfoxino) palladium (II) to form E-hydroxy5-quinoline sulfonic acid, which then coordinates with magnesium to form a fluorescent chelate. Standardization Working standard CN- solutions containing 0.625, 1.25, 2.5, 5.0 and 10.0 pg/ml were prepared fres h weekly by dilution of the stock CN- solution with 0.1 N sodium hydroxide. These standards were assayed each working day. Blood standards w e r e prepared by adding stock CN- solution to a portion of whole blood to give a concentration of 10 g /m1. This preparation was diluted serially with portions of the s a m e blood sample to result in the s a m e concentrations as the aqueous standard. Precision Six concentrations of CN- in 0.1 N sodium hydroxide were assayed 20 times during one working month. One hundred milliliter volumes of each concentration were prepared and stored a t room temperature in glass-stoppered erlenmeyer flasks. Aliquots w e r e withdrawn
BLOOD CYANIDE
165
daily f o r assay. Repeatability was statistically evaluated to determine precision. Correlation
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Fifty s a m p l e s of CN- i n 0.1 N sodium hydroxide w e r e assayed by a manual ( r e f e r e n c e ) method [12] and our automated ( t e s t ) method. E r r o r s of t h e M e t h o d E r r o r s were estimated statistically as outlined by Westgard and Hunt [21], by t h e least-squares p a r a m e t e r s . Random e r r o r was measured by the standard e r r o r of estimate (S ), constant e r r o r by the y intercept (b), and proportional e r r o r by tge slope ( m ) . RESULTS A typical CN- standard c u r v e is shown i n Fig. 3 with concentration v e r s u s fluorescent units plotted on Cartesian coordinate graph paper. The s a m e linear curve is obtained for CN- concentrations up to 10 pg/ml in both 0 . 1 N sodium hydroxide and human whole blood. Therefore, i t is recommended that aqueous standards be used f o r daily calibration because of stability and simplicity of preparation. Precision, expressed as the coefficient of variation in percent, did not exceed * 6.5% f o r aqueous CN- concentrations ranging from 1.3 to 9.8 pg/ml. This figure increased to 11.0% for a CN- concentration of 0.5 Fg/ml. Mean concentrations, standard e r r o r of the mean (SE), standard deviation (SD), and coefficient of variation (CV%) are shown i n Table 1. The reference v e r s u s test correlation of 50 aqueous CN- values was excellent. Plots of the paired data points and regression line are presented in Fig. 4. The least-squares p a r a m e t e r s obtained by statistical evaluation of the correlation data a r e presented in Table 2. From these p a r a m e t e r s , the e s t i m a t e s of proportional, constant, and random e r r o r were S.O%, 0.2 pg/ml, and 0 . 5 pg/ml, respectively. In V i v o M o n i t o r i n g Cyanide concentrations i n the blood of dogs, a f t e r intravenous injection of sodium cyanide, has been continuously monitored by using the in vivo sampler. Figure 5 shows a continuous recording f o r a dog. The initial portion of the c h a r t (right s i d e ) shows the reagent
GROFF E T AL.
166 100 0.1 NORMAL SODIUM HYDROXIDE
90
0
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80
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r'iti. Y. ataaaara c u r v e l o r cyaniae in u. I N soaium nyaroxiae solution and in human whole blood. TABLE 1. Precision of Results f o r Cyanidea Mean 0.49 1.29 3.82 5.49 7.43 9.75
0.054 0.073 0.240 0.3 58 0.472 0.592
SE
cv, %
0.012 0.016 0.054 0.080 0.105 0.132
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5.7 6.3 6.5 6.4 6. 1
BLOOD CYANIDE
167
10-
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FIG. 4. Correlation of reference v e r s u s test cyanide values.
TABLE 2. T e s t and Reference Method Comparison Studies
Cyanide
n
m
b
s y , I.Lg/ml
r
50
0.91
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0.97
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168 GROFF ET AL.
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169
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baseline and control blood baseline. Normal blood gives no i n c r e a s e in reading above the reagent baseline. An initial intravenous injection of 0.1 mg CN- p e r kilogram of body weight was followed a f t e r 16 min by a second injection of 0.2 m g CN- p e r kilogram. CN- peak blood levels, approximately 1 min a f t e r each injection, were 0.9 and I. 9 p g CN-/ml, respectively. Comprehensive studies of cyanide toxicity, metabolism, and therapy will be discussed in a s e p a r a t e report. DISCUSSION The sensitivity of the CN- a s s a y was found to vary directly with changes i n a i r flow r a t e s used to segment the recipient s t r e a m . The volatilization of CN- during the distillation s t e p was facilitated by increasing the air flow rate. Sensitivity a l s o increased as the distillation temperature was increased and a l a r g e r percentage of the cyanide was volatilized. A maximum practical temperature of 95°C was selected. E r r a t i c bubble pattern and flow r a t e exiting from the heating bath, which occurred a t higher temperatures, caused liquid c a r r y o v e r during the vapor phase separation in the C2 connector. Wetting agents, when added to the recipient s t r e a m before entering the heating bath, did not prevent foaming i n the C2 connector. Howe v e r , propanol, added immediately a f t e r distillation, prevented frothing a t 95'C. Octanol, which was quite effective in eliminating foaming, was not used because of rapid dissolution of the Tygon pump tube. Interference from thiocyanate was found to occur when 1.0 N nitric acid was used to acidify the recipient s t r e a m . A thiocyanate solution containing 1 mg/ml gave a reading equivalent to a CN- solution containing approximately 1 pg/ml. This interference, in the presence of strong acid, was due to thiocyanic acid, which would volatilize a t 95°C [ 51. Thiocyanic acid in the vapor s t a t e h a s been reported to be at l e a s t 95% isothiocyanic acid [22]. This interference was eliminated by using 1.0 N acetic acid, thereby raising the pH from 1 to 4. Using the procedure as described in this report, a 1 mg/ml solution of thiocyanate produces no reading. Sulfhydryl compounds and hydrogen sulfide a l s o r e a c t to form the fluorescent chelate and could r e s u l t in a significant level of interference. Protein sulfhydryl compounds are removed by dialysis while diffusable, nonvolatile sulfhydryls are eliminated by distillation. The resulting sensitivity of the automated method ( 0 . 5 pg. m l ) is not as g r e a t a s that of the manual method (0.02 pg/ml) due to inherent l o s s e s during dialysis and distillation, but is s t i l l well below the lethal blood level of 3 pg/ml. If s m a l l readings occur with control blood samples, this should be subtracted from subsequent a s s a y readings.
170
GROFF E T AL.
Any hydrogen sulfide, which would not normally be present in the sample, would be measured as CN-.
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CONCLUSION The reported method is sensitive to 0.5 pg of CN- p e r milliliter of sample material. The assay is specific due to elimination of interfering materials by dialysis and distillation. Precision does not exceed * 6.5% fo r CN- concentrations ranging from 1.3 to 9.8 dml. SUMMARY Discrete blood s am p l es can be assayed a t a r a t e of 20/hr. By means of a double lumen catheter, venous o r a r t e r i a l blood can be monitored continuously. REFERENCES
[41
[ 51
[81 [91
Auto Analyzer Methodology, Bulletin C ~ CCyanide , IVYTechnicon Instruments, Inc., Tarrytown, New York, Auto Analyzer Methodology, Bulletin C7d, Total Cyanide Vb, Technicon Instruments, Inc. , Tarrytown, New York. P. Casapieri, R. Scott, and E. A. Simpson, The determination of cyanide ions in waters and effluents by an auto analyzer procedure, Anal. Chim. Acta, 49, 188 (1970). P. D. Goulden, B. K. Afghan, and P. Brooksbank, Determination of nanogram quantities of simple and complex cyanides in water, Anal. Chem., 44, 1845 (1972). Johns, On the determination of s m a l l A. Viehoever and C. quantities of hydrocyanic acid, J. Am. Chem. SOC., 37, 601 (1915). A. 0. Gettler and L. Coldbaum, Detection and estimation of 270 ( 1947). microquantities of cyanide, Anal. Chem., 3, R. A. Fulton and M. J. Van Dyke, Determination of s m a l l quantities of hydrogen cyanide in insects and plant tissue, Anal. Chem., 19,922 (1947). F. P. Treadwell and W. T. Hall, Analytical Chemistry, Vol. 11: Quantitative Analyses, Wiley, New York, 1935. A. Sieverts and A. Z. Hermsdorf, Der nachweis gasfarmiger 34, 3 (1921). blausPure in luft, Angew. Chem., -
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R. I. Nicholson, The estimation of hydrocyanic a c i d by the phthalin method, Analyst, 66, 189 (1941). ,. I. M. Kolthoff, Uber den nachweis und die bestimmung kleiner mengen cyanwasserstoff, Z. Anal. Chem., 57, 1 (1918). W. N. Aldridge, A new method f o r the estimation of m i c r o quantities of cyanide and thiocyanate, _ Analyst, 69, 262 (1944). __W. N. Aldridge, The estimation of m i c r o quantities of cyanide Analyst, 70, 474 (1945). and thiocyanate, ~R. B. Bruce, J. W. Howard, and R. F. Hanzal, Determination of cyanide, thiocyanate, and alpha-hydroxynitriles in plasma o r s e r u m , Anal. Chem., 27, 1346 (1955). J. Epstein, Estimation o f m i c r o q u a n t i t i e s of cyanide, __ Anal. Chem., - 19, 272 (1947). S. B a a r , The m i c r o determination of cyanide: its application to the analysis of whole blood, 7 -Analyst 91, 268 (1966). W. Konig, b e r eine neue, vom pyridin derivierende k l a s s e von farbstoffen, J. P r a k t . Chem., 69, 105 (1904). J. S. Hanker, R. M. Gamson, and E K l a p p e r , A fluorometric method for estimation of cyanide, Anal. Chem., 29, 879 (1957). J. S. Hanker, A. Gelberg, and B. Witten, Fluorometric and colorimetric estimation of cyanide and sulfide by demasking reactions of palladium chelates, Anal. Chem., 30, 93 ( 1958). W. A. Groff, A. Kaminskis, and S. A. Cucinell, Simultaneous determination of methemoglobin and total hemoglobin by a continuous-flow method, Clin. Chem., 20, 1116 ( 1974). J. W. Westgard and M. R. Hunt, Use a n d i n t e r p r e t a t i o n of common statistical t e s t s in method-comparison studies, Clin. Chem., 19, 49 (1973). C. I. Beard and B. D. Dailey, The s t r u c t u r e and dipole moment 18, 1437 (1950). of isothiocyanic acid, J. Chem. Phys., ~
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A Completely Automated Fluorometric Blood Cyanide Method: A Specific Assay Incorporating Dialysis and Distillation*
WILLIAM A. GROFF,? SAMUEL A. CUCINELL, M. D. ,$ PASQUALE VICARIO, and ANDRIS KAMINSKIS Biomedical Laboratory Edgewood Arsenal Aberdeen Proving Ground, Maryland
*The views of the authors do not purport to reflect the position of the Department of Defense. The use of trade names in this r e p o r t does not constitute an official endorsement o r approval of the u s e of s u c h c o m m e r c i a l hardware o r software. This report may not be cited f o r purposes of advertisement. The volunteers who donated blood for these t e s t s a r e enlisted U.S. Army personnel. These t e s t s are governed by the principles, policies, and rules f o r medical volunteers as established in AR 70-25, and the Declaration of Helsinki. In conducting the r e s e a r c h described in this report, the investigat o r s adhered to the "Guide f o r the C a r e and Use of Laboratory Animals" as promulgated by the Committee on Revision of the Guide f o r Laboratory Animals Facilities and C a r e of the Institute of Laboratory Animal Resources, National Research Council. ?Reprint requests should be a d d r e s s e d to William A. Groff, Biomedical Laboratory, Edgewood Arsenal, APG, Maryland 2 1010. $Present address: Box 0, Gorgas Hospital, Panama Canal Zone. 159 Copyright 0 1977 hy MarLel Dekker. Inc All Rights Reserved Neither this work nor any part may he reproduced or transmitted in any form or by any means. electronic or mechanical. including photocopying, microfilming. and recording, or by any information storage and retrieval system, without permission in writing from the publisher
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160
GROFF E T AL.
The automated blood cyanide method described in this report was developed to meet the requirements of o u r laboratory f o r studies of cyanide poisoning and its therapy. Since existing automated CN- methods [ 1-41 are designed for the a ss ay of water samples, an extensive literature survey was made in o rd er to select the most sensitive and specific method that could be adapted to a practical, automated as s ay of blood CN-. The Prussian blue [5-71, picric acid [8], and copper-benzidine [9] reactions are not sufficiently sensitive to meet our requirements. The o-cresolphthalein [ 101 and phenolphthalein [ 111 methods lack specificity. The methods of Aldridge [12-141 and Epstein [15, 161, based on the Konig [17] reaction, a r e widely used and p o s s e s s adequate sensitivity, but utilize reagents which rapidly deteriorate Tygon plastic tubing. Two fluorometric procedures, one by Hanker, Gamson, and Klapper [ 181 and the other by Hanker, Gelberg, and Witten [ 191, appeared desirable, due to extreme sensitivity and mild reagents. The automation of the latter, with the addition of dialysis and distillation s tep s to eliminate interfering substances, is the subject of this report. M A T E R I A L AND M E T H O D S Reagents 1. Sodium hydroxide, 0.03 N. Dissolve 1.2 gm sodium hydroxide in distilled water. Add 2.0 m l of B r i j 35 and dilute to 1 l i t e r . 2. Sodium hydroxide, 0.5 N. Dissolve 20.0 gm sodium hydroxide in distilled water and dilute to 1 liter. 3. Glycine buffer. Dissolve 77.4 gm glycine and 58.6 gm sodium chloride in distilled water. Add 1.0 ml of Brij 35 and dilute to 1 liter. 4. Palladium chelate, 0.01 gm/liter. Dissolve 0.010 gm potassium b is ( 5-sulfoxino)palladium (11)* in distilled water. Add 1.0 ml B r i j 35 and dilute to 1 liter. The chelate was prepared as follows [ 191 : 8-hydroxy- 5-quinoline sulfonic acid (4.50 gm, 0.02 mole) was added to a solution of palladous chloride (2.14 gm, 0.01 mole) in 300 ml of 5% sulfuric acid. The solution was heated to boiling and then cooled to room temperature. Saturated potassium carbonate solution was added until the evolution of carbon dioxide ceased. The chelate separated as a fine, yellow p r e cipitate. It was collected on a filter, washed successively with 10% potassium carbonate solution, water, alcohol, and ether; then dried in air. *Prepared as described by Hanker et al. [ 191.
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BLOOD CYANIDE
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5. Magnesium chloride, 1.0 gm/liter. Dissolve 1.0 gm magnesium chloride in distilled water. Add 1.0 Brij 35 and dilute to 1 liter. 6. Propanol, 50%. Dilute 500 ml propanol to 1 l i t e r with distilled water. 7. Acetic acid, 1.0 N. Add 60.0 ml concentrated acetic acid to distilled water. Add 2.0 ml Brij 35 and dilute to 1 liter. 8. Heparin solution, approximately 90 units/ml. Add 10.0 ml heparin (1000 units/ml) to 100 ml s t eri l e isotonic saline. 9. Brij 35. Surfactant obtained from Technicon Instruments Corporation, Tarrytown, New York. 10. Stock potassium cyanide solution, 38.4 mmole/liter. Dissolve 0.2 503 gm potassium cyanide in 0.1 N sodium hydroxide and dilute to 100 nil with 0 . 1 N sodium hydroxide. This solution contains 1 mg of CN- per milliliter. Crystalline potassium cyanide is deliquescent and should be stored under d r y conditions. APPARATUS The analytical system consists of the following modules from Technicon Instruments Corporation, Tarrytown, New York: ( a ) s ampler 11, ( b ) model I1 proportioning pump, ( c ) dialyzer (37 "C) with one s e t of dialyzing plates and type C membrane, ( d ) heating bath ( 9 5 ' C ) with a 40-ft glass coil, ( e ) one 20-ft time-delay coil, ( f ) fluorometer In with Corning Glass filters (excitation - #5970 and emission - #4308 and #3389), and ( g ) reco rd e r with percent transmittance paper. PROCEDURE The flow diagram of the automated manifold is shown in Fig. 1 as it is used for intermittent sampling of blood collected by syringe o r Vacutainer. * Sample and water wash cups are placed alternately on the sample platter, which is operated at a ra t e of 40/hr, resulting in a n effective sampling rate of 20/hr. For continuous in vivo monitoring, the Sampler I1 module and intermittent sample pump tube are disconnected. A 0.235 ml/min heparin pump tube and a 0.60 ml/min in vivo sample pump tube a r e attached to the in vivo sampler [20] shown in Fig. 2, using 4-ft lengths of 0.015-in. i.d. Tygon tubing (Technicon stock #116-0536-04). The
*Available from Scientific Products, 8855 McGaw Rd., Columbia, Md. 21045.
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FILTERS E x c i m a n - Cornin$*5970 Emmmn - Corning 4308 and"3389
FLUORONEPHELOMETER 111
TIME DELAY 20 Foot Coil
22:
WASTE
TO SAMPLER I1
FIG. 1. Flow diagram of automated blood cyanide method.
RECORDER
n
PUMPED WAS1 ALIOUOT
TUBE SIZE ML/MINI
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T€CHNICON INSTRUMENTS CORP. CATALOG NO. 116020302 ACOE MEDICAL SUPPLY CATALOG NO. AR814-1%
(c)
(d)
FIG. 2. In vivo sampler for continuous intravenous o r intra-arterial blood sampling.
ALOE MEDICAL SUPPLY CATALOG NO. AR819-1%
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LONGER T H A N INNER CATHETER)
ALOE MEDICAL SUPPLY CATALOG NO. E D 3068
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INNER V C A T H ' b '
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164
GROFF E T AL.
in vivo sample pump tube, which draws a mixture of blood and heparin, is attached to the H3 cactus in place of the intermittent sample pump tube. The blood sample is mixed f i r s t with sodium hydroxide and the erythrocytes completely hemolyzed in a l arge mixing coil before entering the dialyzer. In alkaline solution, CN- is released both from cyanmethemoglobin and from nonspecific binding. The hemolyzate is then dialyzed a t pH 12 and a constant fraction of the CNpresent in the sample s t ream p as s es into the recipient s t r e a m which exits from the dialyzer. The recipient s t r e a m is acidified before entering the 95°C heating bath. During this 50-sec distillation period at pH 4, a constant fraction of the CN- is volatilized into the air bubbles that segment the liquid. This vapor must not come in contact with Tygon plastic tubing, due to reaction and l o s s of the CN-. Therefore, the portion of the manifold from point A to point B on the flow diagram must be constructed of glass-to-glass fittings and connections. The vapor and liquid phases are separated by means of a C2 connector following the addition of propanol to eliminate foaming. A portion of the vapor phase, which is withdrawn from the C2 connector by a difference in flow rates, is used to segment a stream of sodium hydroxide. After the absorption of CN- in alkali, plastic tubing may again be used without l o s s of CN-. An aliquot of the alkali, which contains absorbed CN-, is mixed f i r s t with buffer and then with palladium chelate reagent. CN- demasks the nonfluorescent potassium bis ( 5-sulfoxino) palladium (II) to form E-hydroxy5-quinoline sulfonic acid, which then coordinates with magnesium to form a fluorescent chelate. Standardization Working standard CN- solutions containing 0.625, 1.25, 2.5, 5.0 and 10.0 pg/ml were prepared fres h weekly by dilution of the stock CN- solution with 0.1 N sodium hydroxide. These standards were assayed each working day. Blood standards w e r e prepared by adding stock CN- solution to a portion of whole blood to give a concentration of 10 g /m1. This preparation was diluted serially with portions of the s a m e blood sample to result in the s a m e concentrations as the aqueous standard. Precision Six concentrations of CN- in 0.1 N sodium hydroxide were assayed 20 times during one working month. One hundred milliliter volumes of each concentration were prepared and stored a t room temperature in glass-stoppered erlenmeyer flasks. Aliquots w e r e withdrawn
BLOOD CYANIDE
165
daily f o r assay. Repeatability was statistically evaluated to determine precision. Correlation
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Fifty s a m p l e s of CN- i n 0.1 N sodium hydroxide w e r e assayed by a manual ( r e f e r e n c e ) method [12] and our automated ( t e s t ) method. E r r o r s of t h e M e t h o d E r r o r s were estimated statistically as outlined by Westgard and Hunt [21], by t h e least-squares p a r a m e t e r s . Random e r r o r was measured by the standard e r r o r of estimate (S ), constant e r r o r by the y intercept (b), and proportional e r r o r by tge slope ( m ) . RESULTS A typical CN- standard c u r v e is shown i n Fig. 3 with concentration v e r s u s fluorescent units plotted on Cartesian coordinate graph paper. The s a m e linear curve is obtained for CN- concentrations up to 10 pg/ml in both 0 . 1 N sodium hydroxide and human whole blood. Therefore, i t is recommended that aqueous standards be used f o r daily calibration because of stability and simplicity of preparation. Precision, expressed as the coefficient of variation in percent, did not exceed * 6.5% f o r aqueous CN- concentrations ranging from 1.3 to 9.8 pg/ml. This figure increased to 11.0% for a CN- concentration of 0.5 Fg/ml. Mean concentrations, standard e r r o r of the mean (SE), standard deviation (SD), and coefficient of variation (CV%) are shown i n Table 1. The reference v e r s u s test correlation of 50 aqueous CN- values was excellent. Plots of the paired data points and regression line are presented in Fig. 4. The least-squares p a r a m e t e r s obtained by statistical evaluation of the correlation data a r e presented in Table 2. From these p a r a m e t e r s , the e s t i m a t e s of proportional, constant, and random e r r o r were S.O%, 0.2 pg/ml, and 0 . 5 pg/ml, respectively. In V i v o M o n i t o r i n g Cyanide concentrations i n the blood of dogs, a f t e r intravenous injection of sodium cyanide, has been continuously monitored by using the in vivo sampler. Figure 5 shows a continuous recording f o r a dog. The initial portion of the c h a r t (right s i d e ) shows the reagent
GROFF E T AL.
166 100 0.1 NORMAL SODIUM HYDROXIDE
90
0
HUMAN WHOLE BLOOD
80
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7 8
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91
100
CYANIDE ug/rnl
r'iti. Y. ataaaara c u r v e l o r cyaniae in u. I N soaium nyaroxiae solution and in human whole blood. TABLE 1. Precision of Results f o r Cyanidea Mean 0.49 1.29 3.82 5.49 7.43 9.75
0.054 0.073 0.240 0.3 58 0.472 0.592
SE
cv, %
0.012 0.016 0.054 0.080 0.105 0.132
11.0
a Each concentration was measured 20 times.
5.7 6.3 6.5 6.4 6. 1
BLOOD CYANIDE
167
10-
98-
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REFERENCE METHOD
FIG. 4. Correlation of reference v e r s u s test cyanide values.
TABLE 2. T e s t and Reference Method Comparison Studies
Cyanide
n
m
b
s y , I.Lg/ml
r
50
0.91
0.22
0.518
0.97
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168 GROFF ET AL.
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BLOOD CYANIDE
169
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baseline and control blood baseline. Normal blood gives no i n c r e a s e in reading above the reagent baseline. An initial intravenous injection of 0.1 mg CN- p e r kilogram of body weight was followed a f t e r 16 min by a second injection of 0.2 m g CN- p e r kilogram. CN- peak blood levels, approximately 1 min a f t e r each injection, were 0.9 and I. 9 p g CN-/ml, respectively. Comprehensive studies of cyanide toxicity, metabolism, and therapy will be discussed in a s e p a r a t e report. DISCUSSION The sensitivity of the CN- a s s a y was found to vary directly with changes i n a i r flow r a t e s used to segment the recipient s t r e a m . The volatilization of CN- during the distillation s t e p was facilitated by increasing the air flow rate. Sensitivity a l s o increased as the distillation temperature was increased and a l a r g e r percentage of the cyanide was volatilized. A maximum practical temperature of 95°C was selected. E r r a t i c bubble pattern and flow r a t e exiting from the heating bath, which occurred a t higher temperatures, caused liquid c a r r y o v e r during the vapor phase separation in the C2 connector. Wetting agents, when added to the recipient s t r e a m before entering the heating bath, did not prevent foaming i n the C2 connector. Howe v e r , propanol, added immediately a f t e r distillation, prevented frothing a t 95'C. Octanol, which was quite effective in eliminating foaming, was not used because of rapid dissolution of the Tygon pump tube. Interference from thiocyanate was found to occur when 1.0 N nitric acid was used to acidify the recipient s t r e a m . A thiocyanate solution containing 1 mg/ml gave a reading equivalent to a CN- solution containing approximately 1 pg/ml. This interference, in the presence of strong acid, was due to thiocyanic acid, which would volatilize a t 95°C [ 51. Thiocyanic acid in the vapor s t a t e h a s been reported to be at l e a s t 95% isothiocyanic acid [22]. This interference was eliminated by using 1.0 N acetic acid, thereby raising the pH from 1 to 4. Using the procedure as described in this report, a 1 mg/ml solution of thiocyanate produces no reading. Sulfhydryl compounds and hydrogen sulfide a l s o r e a c t to form the fluorescent chelate and could r e s u l t in a significant level of interference. Protein sulfhydryl compounds are removed by dialysis while diffusable, nonvolatile sulfhydryls are eliminated by distillation. The resulting sensitivity of the automated method ( 0 . 5 pg. m l ) is not as g r e a t a s that of the manual method (0.02 pg/ml) due to inherent l o s s e s during dialysis and distillation, but is s t i l l well below the lethal blood level of 3 pg/ml. If s m a l l readings occur with control blood samples, this should be subtracted from subsequent a s s a y readings.
170
GROFF E T AL.
Any hydrogen sulfide, which would not normally be present in the sample, would be measured as CN-.
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CONCLUSION The reported method is sensitive to 0.5 pg of CN- p e r milliliter of sample material. The assay is specific due to elimination of interfering materials by dialysis and distillation. Precision does not exceed * 6.5% fo r CN- concentrations ranging from 1.3 to 9.8 dml. SUMMARY Discrete blood s am p l es can be assayed a t a r a t e of 20/hr. By means of a double lumen catheter, venous o r a r t e r i a l blood can be monitored continuously. REFERENCES
[41
[ 51
[81 [91
Auto Analyzer Methodology, Bulletin C ~ CCyanide , IVYTechnicon Instruments, Inc., Tarrytown, New York, Auto Analyzer Methodology, Bulletin C7d, Total Cyanide Vb, Technicon Instruments, Inc. , Tarrytown, New York. P. Casapieri, R. Scott, and E. A. Simpson, The determination of cyanide ions in waters and effluents by an auto analyzer procedure, Anal. Chim. Acta, 49, 188 (1970). P. D. Goulden, B. K. Afghan, and P. Brooksbank, Determination of nanogram quantities of simple and complex cyanides in water, Anal. Chem., 44, 1845 (1972). Johns, On the determination of s m a l l A. Viehoever and C. quantities of hydrocyanic acid, J. Am. Chem. SOC., 37, 601 (1915). A. 0. Gettler and L. Coldbaum, Detection and estimation of 270 ( 1947). microquantities of cyanide, Anal. Chem., 3, R. A. Fulton and M. J. Van Dyke, Determination of s m a l l quantities of hydrogen cyanide in insects and plant tissue, Anal. Chem., 19,922 (1947). F. P. Treadwell and W. T. Hall, Analytical Chemistry, Vol. 11: Quantitative Analyses, Wiley, New York, 1935. A. Sieverts and A. Z. Hermsdorf, Der nachweis gasfarmiger 34, 3 (1921). blausPure in luft, Angew. Chem., -
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 12/27/14 For personal use only.
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R. I. Nicholson, The estimation of hydrocyanic a c i d by the phthalin method, Analyst, 66, 189 (1941). ,. I. M. Kolthoff, Uber den nachweis und die bestimmung kleiner mengen cyanwasserstoff, Z. Anal. Chem., 57, 1 (1918). W. N. Aldridge, A new method f o r the estimation of m i c r o quantities of cyanide and thiocyanate, _ Analyst, 69, 262 (1944). __W. N. Aldridge, The estimation of m i c r o quantities of cyanide Analyst, 70, 474 (1945). and thiocyanate, ~R. B. Bruce, J. W. Howard, and R. F. Hanzal, Determination of cyanide, thiocyanate, and alpha-hydroxynitriles in plasma o r s e r u m , Anal. Chem., 27, 1346 (1955). J. Epstein, Estimation o f m i c r o q u a n t i t i e s of cyanide, __ Anal. Chem., - 19, 272 (1947). S. B a a r , The m i c r o determination of cyanide: its application to the analysis of whole blood, 7 -Analyst 91, 268 (1966). W. Konig, b e r eine neue, vom pyridin derivierende k l a s s e von farbstoffen, J. P r a k t . Chem., 69, 105 (1904). J. S. Hanker, R. M. Gamson, and E K l a p p e r , A fluorometric method for estimation of cyanide, Anal. Chem., 29, 879 (1957). J. S. Hanker, A. Gelberg, and B. Witten, Fluorometric and colorimetric estimation of cyanide and sulfide by demasking reactions of palladium chelates, Anal. Chem., 30, 93 ( 1958). W. A. Groff, A. Kaminskis, and S. A. Cucinell, Simultaneous determination of methemoglobin and total hemoglobin by a continuous-flow method, Clin. Chem., 20, 1116 ( 1974). J. W. Westgard and M. R. Hunt, Use a n d i n t e r p r e t a t i o n of common statistical t e s t s in method-comparison studies, Clin. Chem., 19, 49 (1973). C. I. Beard and B. D. Dailey, The s t r u c t u r e and dipole moment 18, 1437 (1950). of isothiocyanic acid, J. Chem. Phys., ~
