15. Manual of Lipid Operations. Lipid Research Clinics Program,
1, Lipid and Lipoprotein Analysis, U.S. Department of HEW, Publication no. (NIH) 75-628, 1974.
16. Allain, C. C., Poon, L. S., Chan, C. S. G., et al., Enzymatic determination of total serum cholesterol. Clin. Chem. 20,470 (1974).
17. Witte, D. L., Barrett, II, D. A., and Wycoff, D. A., Evaluation of
an enzymatic procedure for determination of serum cholesterol with
the Abbott ABA-100. Clin. Chem. 20, 1282 (1974).
18. Steele, B. W., Koehler, D. F., Azar, M. M., eta!., Enzymatic determinations of cholesterol in high-density-lipoprotein
fractions
prepared by a precipitation
technique. Clin. Chem. 22,98 (1976).
19. Liedtke, R.J.,Busby,B.,and Batjer,
J.D.,Use of the Du Pont
aca to measure cholesterol in high-density lipoprotein fractions
prepared by the heparin/Mn2
precipitation method. Clin. Chem.
24, 161 (1978).
20. Warnick, G. R., and Albers, J. J., A comprehensive evaluation of
the heparin-manganese precipitation procedure for estimating high
CLIN.
cholesterol. J Lipid Res. 19, 65 (1978).
J. J., Warnick, G. R., Wiebe, D., eta!., Multi-laboratory
density lipoprotein
21. Albers,
comparison of three heparin_Mn2+ precipitation procedures for estimating cholesterol high-density lipoprotein. Clin. Chem. 24, 853
(1978).
22. Warnick, G. R., and Albers, J. J., Heparin-Mn2
quantitation of
high-density-lipoprotein
cholesterol:
An ultrafiltration procedure for
lipemic samples. Clin. Chem. 24, 900 (1978).
23. Davis, S., and Lanier, J. F., Cholesterol standards-thicker
than
water. Am. Soc. Clin. Pat hol. Summary Report 15 (2): 3 (1978).
24. Nielsen, L. G., and Ash, K. 0., A protocol for the adoption of analytical methods in the clinical chemistry laboratory. Am. J. Med.
Technol. 44, 30 (1978).
25. Castelli, W. P., Cooper, G. R., Doyle, J. T., et al., Distribution of
triglyceride and total LDL and HDL cholesterol in several populations: A cooperative lipoprotein phenotyping study. J. Chronic Dis.
30, 147 (1977).
CHEM.24/12, 2184-2185 (1978)
Analytical Precautions in Measurement of Blood Cyanide
F. Lee Rodkey and Robert F. Robertson
Solutions of KCN in 0.1 mol/liter NaOH, stored for six
months at 4 #{176}C,
were unchanged as determined by silver
titration and color development with pyridine-pyrazalone
reagent. At room temperature the cyanide concentration
of such solutions decreased by 0.079% per day (half life
= 880 days) and the extent of color development changed
in direct proportion to the change in silver titer. Rubber
stoppers adsorb (or dissolve) HCN when in contact with
this gas. The cyanide tends to come out of the stopper long
after the source of the gas is removed.
Rubber stoppers
and expired air contaminated by HCN must be carefully
avoided during cyanide analysis. Expired air contains HCN,
generally in larger amounts for persons who smoke.
We recently published a procedure for measuring blood
cyanide concentration (1). During the development of this
procedure we occasionally observed unexplained, aberrant
results. Further experience with the method has allowed us
to define several additional factors that must be controlled.
These include the stability of stock KCN standard solution
and two sources of cyanide contamination: expired air and
contaminated rubber stoppers.
Materials and Methods
The reagents,
apparatus,and proceduresusedwere exactly
as previously reported (1). Stock KCN solutions were made
Laboratory of Analytical Biochemistry, Naval Medical-Research
Institute, National Naval Medical Center, Bethesda, Md. 20014.
ReceivedAug. 29, 1978;accepted Sept. 18, 1978.
2184
CLINICAL CHEMISTRY,
Vol. 24,
No.12,1978
in 0.1 mol/liter NaOH, prepared with de-ionized water. They
were then stored at different temperatures and in different
containers as noted. Techniques
of silver titration of the stock
KCN solutions and color development
of the working stan-
dards were unchanged from those described
(1).
Results and Discussion
Stability
of KCN: A long-term instability
of stock KCN
solutions in 0.1 mol/liter NaOH was suspected when we analyzed several KCN stock solutions that had been prepared
several months previously and stored at room temperature
without contact with rubber. These solutions gave lower silver-titration values, and the diluted working standards developed less color with the pyridine-pyrazalone
reagent. We
then prepared three stock KCN solutions and analyzed them
periodically
by both the silver titration
and the colorimetric
pyridine-pyrazalone
procedures (Table 1). Stock KCN solutions stored at room temperature
in glass or polyethylene
containers decreased in titer at about the same rate. Only
86.5% of the initial cyanide remained after six months. The
decrease in cyanide at room temperature
follows first-order
kinetics, with a loss of about 0.079% per day-or a half life of
880 days. The sample stored in plastic at refrigerator temperature, 4 #{176}C,
did not change its silver titer by more than
about 2 parts per thousand throughout this entire period.
Each of these solutions was diluted 1000-fold
with 50
mmol/liter
NaOH for colorimetric assay with the pyridinepyrazalone reaction by the routine procedure (1). On the day
the stock solutions were prepared, the absorbance was 0.0228
(range, 0.0227-0.0229) for each nanomole of cyanide for all
solutions. This value remained constant for the stock solution
Table 1. Silver Titration of KCN in 0.1 mol/liter
NaOH, Stored in Glass and Polyethylene
Storage In glass
Days after
KCN, mmol/Ifter
prep.
room temp.
0
15.96
45
15.33
73
15.10
14.95
14.39
13.79
102
137
187
Storage In plastic
uays after
-prep.
-
KCN, mmol/llter
Room temp.
4 #{176}C
no.
Cyanide
recovered,
1
2.7
2
73.9
3
4
5
6’
0.9
23.2
6.6
0.0
nmol
12.61
12.45
12.60
12.59
12.22
12.63
11.76
12.58
11.33
12.59
8Stopper used for cyanmethemoglobin determination on the previous day.
187
10.92
12.63
“New Vacutainer Tubestopper.
CyanIde recovered per minute
Non-smokers
nmol
3.2
3.6
11.2
2.8
stopper
0
28
57
92
142
Table 2. HCN Excreted in Expired Air
Smokers
Table 3. Cyanide Desorbed from Rubber Stoppers
1.6
3.1
0.7
1.0
stored at 4 #{176}C
(range, 0.0227 to 0.0229) throughout
Rubber stoppers: Our laboratory routinely does many total
hemoglobin measurements by the cyanmethemoglobin procedure (3). The solutions are commonly closed with rubber
stoppers (Vacutainer
Tube stoppers) for mixing and to prevent evaporation.
Such rubber stoppers apparently dissolve
HCN derived from the cyanmethemoglobin reagent. Dissolution of the HCN may then contaminate any reagent when
the stopper is used to close another container. The extent of
such contamination
is indicated by the results given in Table
3. We tested a group of five rubber stoppers taken from the
laboratory drawer and one new stopper. The stoppers were
thoroughly washed in detergent and distilled water. Each
stopper was then used to close a glass test tube containing 2
ml of 50 mmol/liter NaOH. The tubes were inverted to allow
the NaOH to remain in contact with the rubber stopper for
the entire
period. The solutions stored at room temperature showed a
progressive loss in color developed that exactly corresponded
48 h. The amount of cyanide present in the NaOH was then
with the decrease in silver titer of the stock solutions. The
measured
colorimetrically.
No cyanide was present in the new
absorbance on day 187 for all three solutions varied from
stopper, but significant
amounts were present in all others.
0.0225 to 0.0230 per nanomole of cyanide, based on the silver
Indeed, the stopper used for a cyanmethemoglobin determination on the previous day provided nearly four times the
titration on that day. These data clearly show that the stock
KCN solutions are much more stable when stored at 4 #{176}C, absorbance
of the standard. The amount of HCN desorbed
from the other stoppers is variable. We now segregate all
there being no detectable loss in six months. Solutions stored
at room temperature
should be regularly re-standardized by
rubber tubing and stoppers used for the cyanide analysis and
silver titration, because the amount of color developed follows
do not use them for any other procedure.
the silver titer.
Effect of expired air: We noted a variation in color develThis work wassupported by the Naval Medical Researchand Development Command, ResearchTask No. MRO41.01.004.0002.The
opment in blanks and standards when certain technicians
opinions and assertions contained herein are the private ones of the
performed the analysis. The problem was finally traced to the
writers and are not to be construed as official or reflecting the views
cyanide present in expired air, which was being absorbed
of the Navy Department
or the naval service at large.
during the pipetting of the alkaline solutions with “blow-out”
Ostwald pipets. The presence of cyanide in expired air was
References
first reported by Boxer and Rickards (2). Our greatest vari1. Rodkey, F. L., and Collison, H. A., Determination
of cyanide and
ability was observed with one technician who smoked. We
nitroprusside
in blood and plasma. Clin. Chem. 23, 1969 (1977).
tested the amount of HCN expired in 1 mm from groups of
2. Boxer, G. E., and Rickards, J. C., Determination
of traces of hylaboratory workers who were smokers and non-smokers. The
drogen cyanide in respiratory air. Arch. Biochem. Biophys. 39, 287
results (Table 2) show that HCN is present in expired air,
(1952).
generally greater for smokers than non-smokers. We now
3. Van Kampen, E. J., and Zijlstra, W. G., Standardization of hemavoid the use of “blow-out” pipets, to eliminate such conoglobinometry. II. The hemiglobincyanide method. Clin. Chim. Acta
tamination.
6,538 (1961).
CLINICALCHEMISTRY,Vol.24,No.12,1978
2185