Fast B test
Fast B is a simple, semi-quantitative method for measuring urinary iodine concentration. Urinary iodine concentration is the most useful test for assessing iodine nutrition in populations. Samples are easy to obtain and over 90% of dietary iodine eventually appears in the urine (1).
ICCIDD/WHO/UNICEF proposed the following classification of iodine nutrition, based on urinary iodine concentration (2):
|200-299||More than adequate
For populations, the most important information is the range in which the median falls
, not the precise value. Therefore, a semi-quantitative method is satisfactory for recognizing the state of iodine nutrition, and therefore, the urgency for action.
The following method - "Fast B" - is adapted from Method B (1,3). In it, urine is digested with ammonium persulfate at 100°C to inactivate impurities and interfering substances (4). Iodine is a catalyst in the Sandell-Kolthoff reaction, in which the yellow-colored ceric ammonium sulfate is reduced to the colorless cerous sulfate, in the presence of arsenious acid (5). The speed of the color change is proportional to the amount of iodine present. Method B used the redox indicator, ferroine, and a stopwatch to measure the speed of color change. Fast B also uses ferroine, but omits the stopwatch and instead groups samples by comparison with reference samples of known iodine content (3).
- Collect about 1-2 ml urine, place in tube and stopper tightly. Samples can be transported at ambient temperature. For storage, it is convenient to refrigerate or freeze, to avoid unpleasant odors, although iodine is stable if tubes are tightly sealed. Collection does not need to be sterile, but avoid contamination with anything containing iodine, like iodized salt or antiseptics.Equipment
- The method uses a heating block (dry bath) with blocks containing 20 tubes each; Pyrex test tubes (13 mm x 100 mm); two fixed volume pipettes (0.5 ml and 1.0 ml); one adjustable pipette (0-200 microliters); and a multipet (Eppendorf) for quick reagent volume additions of 0.125 ml and 0.1 ml.Chemicals
- The following reagents are needed, all analytical grade: potassium iodate; arsenic trioxide, ammonium persulfate (ammonium peroxydisulfate); ammonium cerium (IV) sulfate dihydrate; sodium chloride; ferroine; sulfuric acid. All are generally available from commercial lab supply houses.
1. 114.0 grams ammonium persulfate made up to 500 ml with water; can be stored at room temperature but away from light, stable for at least one month.
2. H2SO4, 5N.
3. Arsenious acid solution - to 10 grams As2O3 and 50 grams NaCl, slowly add 400 mL 5N H2SO4; add water to about 1 liter, heat gently to dissolve (takes several hours), cool to room temperature, dilute with water to two liters, filter, store in a dark bottle away from light at room temperature; stable for at least six months.
4. Sodium chloride, 20% (40 grams in 200 mL).
5. H2SO4, 60% (21.6 N).
6. Ceric ammonium sulfate - dissolve 16 g ceric ammonium sulfate in 1 liter of 2.5 N H2SO4 (2.5 N H2SO4 is made by diluting one part 5 N H2SO4 with one part H2O); solution is stable for more than six months in a dark bottle.
7. Iodine standards
- (a) concentrated iodate solution - 787 mmol/L (100 mg/ml); dissolve 168.5 mg potassium iodate in deionized water, make to 1 liter;
- (b) stock iodate solution, 7.87 mmol/L (1 mg/mL), diluted from (a);
- (c) the stock solution (b) is diluted to make working standards of 0.40 mmol/L (50 mg/L), 0.79 mmol/L (100 mg/L), and 2.37 mmol/L (300 mg/L) in the standard assay; a wide range of other working standard solutions can be prepared if desired.
8. Ferroine/arsenious acid solution - Prepared shortly before use, by mixing 2 mL of 60% H2SO4, 2 mL arsenious acid solution, 4 mL of 20% sodium chloride, and 2 mL ferroine.
Adjustment of conditions
Several parameters were investigated to improve the method previously described (3). Ammonium persulfate was substituted for chloric acid in the digestion step because of the latter's toxicity. Smaller amounts of ammonium persulfate were occasionally associated with inappropriate color change and an insoluble yellow precipitate; increasing the amount of added ammonium persulfate corrected these problems. Allowing the color change to develop more slowly permitted the inclusion of more tubes in each run. Several different sample volumes were also tried, to achieve linearity up to 2.37 mmol/L (300 mg/L). For some, but not all samples, different volumes gave discordant values, usually too low; this probably reflected the continued presence of unidentified interfering substances. Volumes of 150 microliters or less showed no inhibition in any of the samples measured under our digestion conditions.
The following is the procedure as finally developed:
1. Heat each tube, containing 0.15 ml of urine or of standard, and 1.0 ml ammonium persulfate solution, for one hour in the block at 100°C.
2. After cooling at room temperature, add 0.5 ml arsenious acid solution to each tube, and mix on a vortex.
3. At least 15 minutes later, add 0.125 ml of fresh ferroine-arsenious acid solution.
4. Mix tubes on a vortex and range in racks as follows: first, three standards (0.40 mmol/L (50 mg/L), 0.79 mmol/L (100 mg/L), and 2.37 mmol/L (300 mg/L)), then the urine samples and controls, and at the end, a second set of the same three standards.
5. A total of 45-55 tubes - samples, blanks, and controls - are included in each batch.
6. To each tube rapidly add 0.1 ml ceric ammonium sulfate solution with the multipipetter, with quick shaking of each rack, and watch all tubes closely.
7. After an initial blue color, samples turn first to a purple color, later to an orange/brown. The speed of the color change depends on the iodine concentration. As each tube turns purple, place it in another rack in order of color change following addition of the ceric ammonium sulfate. Thus, all tubes, standards, and samples, are now ranked in order of color change.
8. Next count the number of samples falling into each of the four categories (>2.37 mmol/L (>300 mg/L), 0.79-2.37 mmol/L (100-300 mg/L), 0.40-0.79 mmol/L (50-100 mg/L), and <0.40 mmol/L (< 50 mg/L)), from each tube's position relative to those of the standards.
9. Interpret results as follows:
|<50 µg/L||Deficient (moderate/severe)|Notes on procedure
Under these conditions, samples with >2.37 mmol/L (>300 mg/L) change color in less than two minutes, 2.37 mmol/L (300 mg/L) at about two minutes, 0.79 mmol/L (100 mg/L) at about five minutes, 0.40 mmol/L (50 mg/L) at about ten minutes, and 0.08 mmol/L (10 mg/L) at about 40 minutes. For most purposes, it is satisfactory simply to record the number that have not changed before the 0.49 mmol/L (50 mg/L) standard.
Each assay run needs its own standards, so having the maximum number of samples in a given run is desirable. The limits are set by the speed of the color change and the number of tubes that can be watched at one time - about 45 tubes, including 39 unknown samples.
This description focuses on standards of 2.37, 0.79, and 0.40 mmol/L (300, 100, and 50 mg/L), to conform to the recommended categories for defining iodine nutrition (1). Any other standards can be used to define other ranges of interest.
Samples gave values in the appropriate ranges when diluted or when spiked with iodide. Addition of thiocyanate, ascorbic acid, or glucose did not affect the values.Other comments
It is estimated that one technician can easily measure 200 samples in a working day. The cost is low, especially if personnel salaries are not high. The method is easy to learn; e.g., two technicians from a developing country in Africa became proficient in the method after three days of instruction and practice.SourceGnat D, Dunn AD, Chaker S, Delange F, Vertongen F, Dunn JT. Fast colorimetric method for measuring urinary iodine. Clin Chem 2003; 49: 186-188
Method developed by ICCIDD, with funding from the Micronutrient Initiative. For further information, contact: Daniella_GNAT@stpierre-bru.be
1. Dunn JT, Crutchfield HE, Gutekunst R, Dunn AD. Methods for Measuring Iodine in Urine. The Netherlands: International Council for Control of Iodine Deficiency Disorders, 1993.
2. ICCIDD, WHO, UNICEF. Assessment of Iodine Deficiency Disorders and Monitoring their Elimination. A guide for programme managers, 2nd ed. World Health Organization, 2001.
3. Dunn JT, Myers HE, Dunn AD. Simple method for assessing urinary iodine, including preliminary description of a new rapid technique ("Fast B"). Exp Clin Endocrinol Diabetes 1998; 106(suppl 3):1005-1007.
4. Pino S, Fang SL, Braverman LE. Ammonium persulfate: a safe alternative oxidizing reagent for measuring urinary iodine. Clin Chem 1996; 42:239-243.
5. Sandell EB, Kolthoff IM. Micro determination of iodine by a catalytic method. Mikrochemica Acta 1937; 1:9-25.