Application of Pelletized Sodium Borohydride in the

Journal of Analytical Toxicology, Vol. 6, November/December 1982

Application of Pelletized Sodium Borohydride in the Spectrophotometric Determination of Arsenic M. Lopez-Rivadulla, P. Fernandez, A. Carracedo, L. Concheiro, J.R. Alonso* and A. Concheiro** Department of Legal Medicine, Toxicology Service, Pediatric Department*, Pharmaceutical Technical Department**, University of Santiago de Compostela, Spain,

ly requirements being appropriate glassware and a conventional visible spectrophotometer (6). The disadvantage of this type of procedure is the large number of reagents required; which, from the analytical point of view, is a considerable inconvenience.

Materials and Methods Reagents

Introduction The classic method for the detection and determination of arsenic (1) is based on the formation of hydrogen arsenide (arsine) from nascent hydrogen liberated in solution by suitable reagents. Metal complex with silver diethyldithiocarbamate, Ag(DDC), exhibited good stability and these properties rendered them suitable for quantitative analysis (2). Vasak and Sedivec (3) pioneered the use of Ag(DDC) to form a complex with the arsine produced through the Gutzeit technique. Others (4) have modified the composition of the reagents, notably the pyridine solution of Ag(DDC); due to its special characteristics, the analysis was difficult. Atomic absorption spectrophotometry (AAS) constituted an excellent analytical procedure for the determination of toxic metals, but had certain disadvantages; the most important of which were high cost and limited use in forensic toxicology, where it was applicable only for the determination of arsenic (5). However, this technique could also be used for the control of metallic contaminants in the wider context of environmental toxicology. For these reasons, it was difficult to justify the use of AAS for routine work in purely forensic toxicology labs, which requires a simple yet low cost analytical method. A colorimetric method was therefore chosen in which arsenic, in the form of arsine, forms a complex with Ag(DDC); the on314

Arsenic trichloride standard solution (1000 #g/mL) was obtained from BDH Chemicals. Hydrochloric acid solution (37070), concentrated nitric and sulphuric acids, perchloric acid solution (40~ lead acetate, brucine, and silver diethyldithiocarbamate were obtained from Merck. Chloroform (analytical grade) was obtained from Fluka. The silver diethyldithiocarbamate chloroformic solution was made from 2.5 g Ag(DDC) and 0.215 g of burcine, which were dissolved in 1000 mL chloroform. The solution was stored in a tightly stoppered amber bottle, and will be stable if kept refrigerated.

Pellet Manufacture Phosphate was chosen as an excipient because of its noninterfering properties and also because of its stability in concentrated acids, which made it more suitable than organic excipients. The talc was added to help prevent the powder from adhering to the pellet mold. The powdered mixture of 50~ sodium borohydride, 45~ dry calcium phosphate, and 5~ talc was completely mixed and compressed using a rotary type pellet machine (Masque) with a 12ram diameter mold. The hardness of the pellets was determined by a Monsanto hardness tester to be greater than 5 kg. Each pellet weighed 525 mg.

Experimental Arsine (ASH3) was formed from a standard solution of arsenic (II1) by the addition of sodium borohydride, which liberated the H2 in acidic medium. The arsine was made to bubble through

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Journal of Analytical Toxicology, Vol. 6, November/December 1982 the H2 in acidic medium. The arsine was made to bubble through a chloroform solution of Ag(DDC) to obtain the red complex. Standard solutions of arsenic were prepared containing 2, 5, 10, 15, 20, 30, 40, and 5 0 / z g / m L of aqueous solution, and transferred to a volumetric flask of the arsine generator. Five milliliters of chloroform solution, containing 2.5 g Ag(DDC) and 0.215 g of brucine per 1000 mL of chloroform were added to the U-tube. A piece of cotton wool impregnated with lead acetate was placed in the middle section of the U-tube to retain and prevent interference from other hydrides (i.e., H2S, etc.), which may appear during the reaction process. To each of the solutions, 30mL of I N HCI and two 525 mg pellets of sodium borohydride were added and the apparatus was then hermetically sealed. After a few seconds, the arsine bubbled in the U-tube in the proportion to the rate at which the borohydride in the acidic medium liberated the H~. The reaction was completed within 30 to 40 minutes, depending on the amount of arsenic present in the sample. The disintegration time of the pellet was 30 minutes. After the reaction process, the absorption spectrum of the red complex was determined, as shown in Figure 1. This spectrum had two absorption peaks: one at 410 nm and the other at 525 nm. The former was that given by the chloroform solution of Ag(DDC) and the latter only appeared when the complex was formed with arsenic. The working wavelength was 525 rim, where the absorbance of the different concentrations of arsenic was measured.

ture was allowed to stand for a few minutes and then heated gently. The heat was increased to digest the sample until it just began to char, at which stage more nitric acid was added. Charring carries the possibility of reducing arsenic to the trivalent ion and its loss by volatility. The flask contents were allowed to cool to room temperature; 20 m L of nitric acid was added, and 2 mL of perchloric acid. The flask was reheated until white fumes began to appear. The digest should be straw-colored; if not, nitric and perchloric acids were added with further heating until the solution cleared. After the digest was cooled, it was quantitatively transferred to a volumetric flask, then diluted with distilled water. The total time needed to digest a biological sample was approximately 3 to 6 hours. Suitable aliquots of this digest, containing between 3 and 40

A

t 1,C

0.5

~o

~b

Results From the results obtained, the calibration curve (Figure 2) was determined from the following equation:

,~o

3'o

5'0

pg/rnt.

Figure 2. Calibrationcurve for As concentration.

Y = 0.023X + 0.034 Correlation coefficient = 0.997

"/,ABE 9( ......-

Following previously published procedures (7,8), the optimum interval of application was studied and found to be 3 to 40 pg/mL of arsenic (Figure 3). The between-run precision for three different concentrations of arsenic are listed in Table 1.

Application to Biological Samples Two grams of liver, 5 g kidney, 5 mL blood, or 10 mL urine were mixed with 25 mL of concentrated nitric acid and 5 mL of concentrated sulphuric acid in a Kjeldahl flask. The mix-

t

A 0.1

~'5

1.0

115

tog C

Figure 3.

o.:

0.1

o.:

Table I. Between-Run Precision of Method o.:

Aqueous Specimen

o.;

;k

Figure 1. Absorption spectrum of red complex.

t~glmL

Numberof Samples

Mean

SD

CV (%)

5 10 30

10 10 10

5.214 10.15 30.32

0.0994 0.196 0.241

1.9 193 0.80

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Journal of Analytical Toxicology, Vol. 6, N o v e m b e r / D e c e m b e r 1982

#g/mL arsenic, were then assayed. This aliquiot was added to the reaction flask of the arsine generator with 5 mL of 1 N HCI and two 525 mg pellets of sodium borohydride. The apparatus was hermetically sealed, and after a few seconds, the arsine started to bubble in the U-tube. The experimental animal study was carried out with the same procedure. The rabbits had been given oral doses of As20,, the results are shown in Table II.

Table II. Arsenic Concentrations in Rabbit Samples Rabbit Number

Dosage (mglkg)

Liver (g)

Kidney (g)

Blood (mL)

Urine (mL)

1 2 3

50 100 250

3.6 6.3 152

1.82 4.1 8.0

NO 1.26 3.4

NA NA 5.4

ND: Not detectable NA: Not available

Discussion

and Conclusion

From a theoretical point of view, the use of sodium borohydride in the spectrophotometric determination of arsenic presented two main advantages: As a strong reducing agent, it was important in the analysis of biological samples; where, after the destruction and oxidation of organic matter, the arsenic was found in its oxidized form as As(V) and had to be reduced to As(Ill). Due to the chemical properties of sodium borohydride, this step did not present any difficulty. Secondly, the H2 produced in the acidic medium forms arsine with the As(Ill), which bubbled in the chloroform solution of Ag(DDC), thus forming a complex with the arsenic. However, in practice, the reaction of sodium borohydride is so rapid that when used in the classical apparatus, the chloroform solution of Ag(DDC) was expelled and the process invalidated. Others (9) have modified the design of the apparatus to control the bubbling of the acid to a neutral solution that contained sodium borohydride and As. This procedure could not be applied to the analysis of As in biological samples due to its previous oxidation with strong acids, which caused the As to be in acidic medium and in its oxidized state as As(V). In this situation, the addition to the solution of pure sodium borohydride caused the violent production of H., and arsine. It was with these considerations in mind that experimentation was performed, limiting the sodium borohydride to an amount and form sufficient to fulfill its function. For this purpose, the pellet form was found to be ideal, with an optimum weight of 525 mg and a composition of 50% NaBH+, 45% calcium phosphate, and 5% talc. During the analytical process, it was observed that not only was the reaction non-violent but, on the contrary, there was a gradual formation of arsine and hydrogen which obviated the need for any modification of the classical apparatus. The spectrum of the complex corresponded with As(DDC), and from the experimental results obtained, the method could be used for the determination of arsenic in biological samples. A comparison of the reduction procedures is shown in Figure 4, where it can be seen that the classical method (A) was both time consuming and used an excessive number of reagents. The apparatus shown in Figure 4B is a modified version of the classical one. Its main disadvantage lies in the violent reaction caused by the addition of sodium borohydride in a pure state to acidic sample solution. This may be rectified by neutralizing the sample with OHNa, but involves the use of yet another reagent. The method proposed in this paper provides a simple procedure which may be used in any forensic toxicology laboratory for the determination of arsenic in biological samples.

Figure 4. A) Classicalmethod using an excessivenumber of reagents (CuS04, Kl, SnCI2, HCI, Zn. B) Modified design of limited use for biological samples, using HCI and NaBH4 pure as reagents. C) Modified version used herein with no limitations in its application to biological samples, using HCI and NaBH4in pellets with excipients.

References 1. C.P. Stewart and A. Stolman (Eds.). In Toxicology: Mechanisms and Analytical Methods. Volume I1. Academic Press New YorkLondon, 1961, pp. 647-48. 2. A. Wyttenbach and S. Bajo. Extraction with metal-dithiocarbamates as reagents: Anal. Chem. 47(11): 1813-17 (1975). 3. V. Vasak and V. Sedivec. Colorimetric determination of arsenic. Chem. Listy. 48:341 (1952). 4. H. Bode and K. Hachmann. Zur photometrischen arsenbestimmung mit silber-diathyldithiocarbamidat. Z. Anal Chem. Vol. 229: 261-66 (1967). 5. R.C. Baselt and R.H. Cravey. In Casarett and Doull's Toxicology. J. Doull, C.D. Klaasen, and M.S. Amdur (Eds.). Second Edition. Macmillan Publishing Co., Inc. New York. 1980. Chapter 26, p. 66. 6. I. Sunshine. Methodology for Analytical Toxicology. CRC Press Cleveland Ohio, 1975, pp. 31-33. 7. C.G. Cannon and J.S.G. Butterworth. Beer's Law and spectrophotometer linearity. Anal. Chem. 25:168-70 (1953). 8. A. Ringbom. Uber die genauigkeit der colorimetrischen analusen methoden. Z. Anal. Chem. 115:332 (1938). 9. M.I. Basadre, A. Alvarez, F. Bermejo, and M.H. Bollain. Spectrophotometric determination of arsenic with diethyldithiocarbamic acid silver salt and borohydride as a reducing agent. Microchem. J. 23:360-65 (1978).

Manuscript received February 24, 1982; revision received August 6, 1982.

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