4- 18 C 4- C 0 0 U C 18 U I- U 0’ 0 100 200 300 764 CLINICAL CHEMISTRY, Vol. 41, No. 5, 1995 rus, and parathyroid hormone interrela- tionships in pregnancy and newborn infants. Obstet Gynecol 1977;50:701-5. 4. Pitkin RM, Cruikshank DP, Schau- berger CW, Reynolds A, Williams GA, Hargis GK. Fetal calcitropic hormones and neonatal calcium homeostasis. Pediatrics 1980;66:77-82. 5. Steichen JJ, Tsang RC, Gratton TL, Hainstra A, DeLuca HF. Vitamin D ho- meostasis in the perinatal period: 1,25-dihy- droxyvitainin D in maternal, cord and neo- natal blood. N Engl J Med 1980;302:315-9. 6. Wandrup J, Kjer JJ, Thode J, Siggaard- Andersen 0. The concentration of free calcium ions and total calcium in pregnancies at term. ScandJ Clin Lab Invest 1982;42:273-7. 7. Wandrup J. Critical analytical and clin- ical aspects of ionized calcium in neonates. Chin Chem 1989;35:2027-33. Martin Rudnicki’ Jan Stener J#{248}rgensen Dept. of Obstet. and Gynecol. Hvidovre Hospital University of Copenhagen Copenhagen, Denmark ‘Mdress for correspondence: Dept. of Ob- stet. and Gynecol., Rigshospitalet, University Hospital, DK-2100 Copenhagen, Denmark. Alkaline Azobilirubin Color Reaction to Determine Sodium Azide To the Editor: The interference of sodium azide in the determination of bilirubin by diazo- tized sulfanilic acid (Ehrlich’s reagent) has been well documented (1-3). How- ever, because of its bacteriostatic prop- erty and unawareness of interference from sodium azide in the measurement of several constituents of biological flu- ids by use of certain reagents, this pre- servative is still frequently used in var- ious biological samples. We have seen a substantial de- crease in the color yield of the alka- line azobilirubin color reaction in the presence of sodium azide (4). Azide inhibits the formation of azopigment in the reaction between bilirubin and diazotized sulfanilic acid. However, this inhibition is not specific for azide, and ascorbic acid (e.g.) has a similar effect on this reaction. Recently we detected sodium azide in our bilirubin-erythrocyte binding assay, in which albumin samples (containing 0.2 g/L sodium azide as preservative) used for bilirubin ex- traction from erythrocytes interfered in the color reaction. Extending these studies to determine the effect of so- dium azide on the alkaline azobiliru- bin color reaction for bilirubin deter- mination, we found a linear decrease in color yield on increasing azide con- centration. We thus used this finding for the determination of azide concen- trations in biological samples and, as we expected, the method worked well. A standard bilirubin (Sisco Re- search Labs., Bombay, India) solution (74 mgldL or 1.27 mmolIL) was pre- pared as described by Bratlid (5), and its concentration was determined by the method of Fog (6). A stock solu- tion (0.8 g/L) of sodium azide (SD’s Fine Chemicals, Bombay, India) was prepared in the same solution. Fog’s reagent I (caffeine reagent), reagent II (diazo reagent), and reagent III (alkaline tartrate) were prepared as described by Fog (6). The method is as follows: To 75- 313 L of solution containing 60 -250 p.g of sodium azide, we added 100 L of 1.27 mmoIIL bilirubin solution and brought the volume to 1.0 mL with 38 mmoIfL sodium carbonate solution containing 5 mmo]/L EDTA, pH 11.0 (use of 0.07 mohJL sodium phosphate buffer, pH 8.0, instead of sodium car- bonate solution did not affect the color intensity). Then we added 2 mL of Fog’s reagent I, followed by 0.5 mL of reagent II, and mixed well. After 10 min of incubation at room tempera- ture, we added 1.5 mL of Fog’s re- agent III and recorded the absorbance at 600 nm. The percent decrease in the color intensity of bilirubin in the presence of sodium azide was calcu- lated as [(A1 - A2)/A1] x 100, where A1 and A2 are the absorbances in the absence and the presence of sodium azide, respectively. A plot of the amount of azide (with- in the range 60-250 g) vs percent decrease in color intensity (Fig. 1) showed a linear relation. When we used two different biirubin concen- trations (56 and 127 moVL), the re- sults fit the following linear equa- tions, respectively: % decrease in color = [0.55 sodium azide (pg)] - 33.6 % decrease in color = [0.41 sodium azide (pg)] - 28.4 Because the range of linearity of the curve varied at different bilirubin con- centrations, we used the curve obtained with the lower concentration (56 pmoIiL) of bilirubin to determine aside concentration in the range 60-200 tg, and the curve obtained with 127 molJL bilirubin for aside in the range 70-250 .tg. In fact, we found the biliru- bin concentration of 127 p.mol/L to be more appropriate for determining the concentration of sodium azide in vari- ous biological samples. Bilirubin at Sodium azidi(p.) Fig. 1. Dependence of decrease in color intensity (%) on sodium aside concentra- tion, as determined with bilirubin concentra- tions of 127 (#{149}) and 56 ,moVL (0). concentrations <56 p.moWL and >127 JLmoI/L did not yield good results, ow- ing to either deviation from linearity or high color intensity in the control. To use this method with biological sam- ples already containing bilirubin, we recommend exposing the samples to light to degrade the bilirubin already present. References 1. Khayain-Bashi H, Sims C. Effects of sodium azide on the quantitation of chem- ical constituents of serum. Am J Chin Pathol 1978;69:405-9. 2. Eckfeldt J, Freier E, Leiendecker C. Azide interference with bilirubin proce- dures using diazotized sulfaniic acid (Ehrlich’s reagent) [Letter]. Chin Chem 1978;24:2071-2. 3. Khayam-Bashi H. More on interference of sodium azide with measurement of total and conjugated bihirubin in serum [Let- ter]. Chin Chem 1979;25:489. 4. Tayyab 5, Mi MK. Interference of so- dium aside with the quantitation of biliru- bin: modification of Fog’s method to elim- inate aside interference. Anal Biochem 1995;224:542-6. 5. Bratlid D. Bilirubin binding by human erythrocytes. Scand J Clin Lab Invest 1972;29:91-7. 6. Fog J. Determination of bihirubin in serum as alkaline azobihirubin. Scand J Clin Lab Invest 1958;10:241-5. Sand Tayyab1 Mohammad K. All Protein & Enzyme Lab. Interdisciplinary Biotechnol. Unit Aligarh Muslim Univ. Aligarh - 202 002 U.P., India 1 Author for correspondence.