Elemental speciation for chromium in chromium picolinate products

.•

SPECTROCHIMICA ACTA PART B

SpectrochimicaActa Part B 51 (1996) 1801-1812

ELSEVIER

Elemental speciation for chromium in chromium picolinate products 1'2 H o n g D i n g , L i s a K. O l s o n , J o s e p h A. C a r u s o * Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA

Received 12 February1996;accepted 17 May 1996

Abstract

Chromium picolinate products have been examined for different forms of chromium, using chromatographic separation and inductively coupled plasma mass spectrometric detection. The brands we evaluated contained no detectable amount of elemental chromium(VI), the toxic form. Since chromium picolinate might have other chromium forms as impurities, different products may contain different forms of chromium species. Compared with ion-exchange, reversed-phase chromatography showed excellent chromium recovery based on the amount stated on the product label. Keywords: Chromium picolinate; Elemental speciation; Inductively coupled plasma; Ion-exchange chromatography; Reversed-

phase chromatography

1. Introduction

Deficiencies of certain essential elements in the diet are known to contribute to certain diseases. Chromium is one element that has been implicated [1-5]. It has been suggested as a cofactor in the maintenance of normal lipid and carbohydrate metabolism by assisting the action of insulin [6,7]. Chromium deficiency can be a result of diets low in biologically available chromium, age, pregnancy, high glucose intake, and stress [8-12]. Chromium deficiency in humans and animals leads to impaired glucose tolerance, elevated blood glucose levels, hypercholesterolemia, and development of aortic plaques [13,14]. Carefully conducted studies have shown that patients with coronary artery disease had lower serum chromium concentrations than subjects with normal * Correspondingauthor. 1Paper presented in part at the Pittsburgh Conference, New Orleans, LA, USA, March6-10, 1995. 2This paperwas publishedin the SpecialIssue of Spectrochimica Acta, Part B, devotedto FlowAnalysis.

arteries [4]. A chromium supplement plays an essential nutritional role and can help reduce or prevent the above clinical symptoms. Indeed, chromium supplements, with organic chromium complexes being most effective, have been used widely to aid the treatment of diabetes and high cholesterol levels. In 1980, the National Research Council and the National Academy of Sciences recommended a daily intake of chromium of 50-200/~g [15]. Picolinic acid is an isomer of nicotinic acid, which is a natural metabolite of tryptophan produced in the liver and kidney. It is an effective metal chelator that forms sturdy, acid-stable chelates with transition metals such as zinc and chromium. Chromium picolinate consists of three molecules of picolinic acid and one trivalent chromium atom. This stable complex is electrically neutral and relatively lipophilic, thus, it is expected to aid the absorption and intraceUular uptake of chromium. This superior permeability to cell membranes allows more effective enhancement of insulin activity at modest doses [16]. It can therefore provide a practical and safe alternative chromium supplement.

0584-8547/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PI1 S0584-8547(96)01549-2

H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

1802 Table 1 ICP operating conditions Forward power Reflected power Nebulizer gas flow O2 flow Coolant gas flow Auxiliary gas flow Single ion monitoring

Table 3 Cr recovery (%) vs. different incubation conditions 15.5 kW 7W 0.708 1 min -1 0.102 1 rain -t 14 1 min -1 0.8 1 min -1 m/z 53

Table 2 Cr concentration and recovery for different amount of available solvent

1 tablet/320 ml 1 tablet/160 ml 1 tablet/80 ml 1 tablet/40 ml 1 tablet/20 ml 1 tablet/10 mi 1 tablet/5 ml 1 tablet/2.5 ml

Labeled value/ ppm

ICP data/ ppm

Recovery by ICP/%

% RSD ~

0.63 1.3 2.5 5 10 20 40 80

0.59 1.2 2.3 4.4 9 17 32 56

95 96 92 88 90 85 80 70

-

Neutral pH Room temperature

Acidified (pH 3) 37°C

1 2 3 4 18

104 94 92 96 99

92 93 95 94 100

Table 4 Cr recovery after 16 h incubation time

3 8.7 15 -

Trial

Neutral pH Room temperature 6 h incubation time

Acidic, pH 2 37°C 16 h incubation time

1 2 3 4 5 Average

92 93 96 95 94 94

93 94 98 99 97 96 2.7

% RSD

a Based on filtrates of five different trials,

9O

Incubation time/h

1.7

Cr(IID-EDTA

8O 7O

50

40

Cr(Vl)

30 2O 10

0 0

1

2

3

4

T~

5

6

7

8

9

(~)

Fig. 1. Separation of 100 ppb (as Cr) standard mixture of Cr(III)-EDTA and Cr(VI). Ion-exchange column, 50 mM ammonium sulfate buffer, pH 10, adjusted with ammonium hydroxide. Flow rate 2 ml min -1. Injection volume 100 #l. ICP-MS m/z 53.

H. Ding et aL/Spectrochimica Acta Part B 51 (1996) 1801-1812

heavily promoted ingredient in many dietary products that contribute to weight loss. However, there are no FDA regulations on this over-the-counter substance. So far, only mild gastrointestinal disturbances have been reported [6,7]. However, acute and adverse symptoms experienced by certain patients should cause concern [18]. The trivalent chromium is essential while the hexavalent is highly toxic. Thus, importance and toxicity are not only functions of dosage, but also of the chemical form in which the element occurs. Therefore, it is of interest to evaluate chromium species in chromium picolinate-containing products to ascertain whether potentially dangerous species are present. This problem has been addressed by others [19] by analyzing the acid digestion products of several mineral supplements. Our approach is speciate different chromium forms present in the commercial products utilizing mild sample preparation conditions, thus preserving the species' integrity.

Table 5 Chromatographic conditions of ion-exchange separation Guard column/ analytical column

Dionex NG1/Dionex AS 7 anion-exchange

Buffer Buffer pH Flow rate Injection volume

50 mM (NH4)2SO4 9.2 (adjusted with NH4OH) 2 ml min -1 100/zl

1803

The commonly-mentioned chromium picolinate actually refers to chromium tripicolinate, because the mono- and dipicolinate complexes appear to be far less effective in promoting insulin activity in vitro [17]. Recently, chromium picolinate has attracted a lot of attention, not only because it is a much better chromium supplement than other norganic (chromic chloride) and organic chromium complexes (chromium dinicotinate), but also because it appears to assist in weight loss. It is believed to help reduce body fat and increase lean muscle size and strength, and is widely used by professional and amateur body builders in their training regime. Its popularity is evident by its overflowing presence in the health food market and it has rapidly become a

2. Instrumentation and reagents A Dionex AS-450 HPLC (Dionex, Sunnyvale, CA,

40 2 35

T w o forms of Cr

I

30

15

0

I

2

3

4

5

6

7

Time (rain) Fig. 2. Analysis of chromium picolinate filtrate freshly prepared by dissolving one tablet of commercial product in 20 ml water. LC conditions are the same as those in Fig. 1.

1804

H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

rOil)-EIYI'A

30

it

25

15 5 0

0

1

Time (rain)

25 20

10

0

1

2

3

4 5 6 Time (rain)

7

8

9

10

Fig. 3. Injection of chromium picolinate filtrate, as in Fig. 2. (a) Filtrate mixed with an equal volume of 100 ppb Cr(III)-EDTA. (b) Filtrate mixed with 500 ppb Cr(VI), 4/1 (v/v).

USA) system was used, which is equipped with an online degassing system and UV-VIS detector. A Fison PQII + Turbo ICP mass spectrometer (VG Elemental, Winsford, UK) was later coupled to the HPLC system for trace level analysis. The inductively coupled plasma-mass spectrometry (ICP-MS) operating conditions are listed in Table 1. Anion-exchange chromatography is described in detail elsewhere [20]. Briefly, the mobile phase was prepared from ammonium sulfate, doubly-distilled ammonium hydroxide (both from Aldrich, Milwaukee, WI, USA) and distilled, deionized water of 18 Mf~ quality (Barnstead, Boston, MA, USA). A Dionex AS7 anion-exchange column along with an NG1 guard column were used. Reversed-phase chromatography was explored later in the study and a Baxter (Muskegon, MI, USA) OD5, C-18 column was chosen for this separation. HPLC grade methanol (Sigma, St. Louis, MO, USA) was mixed with distilled, deionized water (v/v) for

preparation of the mobile phase for reversed-phase studies. The mobile phases were later filtered through a 0.45 #m nylon filter (Alltech, Deerfield, IL, USA) and degassed on-line with He prior to use. Stock solutions of 500 ppm Cr(III) and Cr(VI) were prepared by dissolving CrCI3-6H20 and K2Cr207 (Fisher, Fair Lawn, N J, USA) in water, respectively, and were diluted to the required concentrations daily before use. Chromium picolinate tablets (Nutrition 21, San Diego, CA, USA) were ground, dissolved in water, and later filtered through a 0.45 #m syringe filter (Alltech). The disodium salt of EDTA (Fisher) was used for chelation of Cr(III). Simple chelation was carried out by heating a mixture of 0.256 g CrC13.6H20, 7.7g EDTA and 100 ml distilled, deionized water [21] at 50°C for 3 h. Chelation of chromium picolinate samples with EDTA was performed in the same fashion, the ratio of EDTA to the amount of chromium in the filtrate being 30:1 (wt/wt).

H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

1805

20

15

0

L)

10

o 0

1

2

3

4

$

Time (rain) Fig. 4. Analysisof synthesizedchromiumpicolinateusingion-exchangechromatography.Sampleconcentration40 ppm. LC conditionsare the same as in Fig. 1.

3. Results and discussion

3.1. Total analysis Total chromium concentrations were determined by direct nebulization of prepared sample solutions using ICP-MS detection.

3.1.1. Chromium recovery with different amounts of solvent One tablet ( ~ 0.55g) of chromium picolinate was dissolved in different amount of water (320-2.5 ml). All samples were analyzed after standing for a 24 h period at room temperature. Table 2 shows a comparison of chromium contents labeled by the manufacturer with those determined by this experiment. Recoveries decreased with decreasing amount of available solvent. Thus, it is recommended that each tablet should be dissolved in at least 20 ml water to ensure complete solubilization of chromium. 3.1.2. Effect of incubation conditions Two sets of samples were prepared in water different lengths of time before being filtered analysis. The first set was prepared by dissolving ground powder of half a tablet (~0.275 g)

for for the of

chromium picolinate in 160 ml water (neutral pH), and allowing to stand at room temperature for certain time periods before analysis. The second set was prepared in pH 3 water (acidified with HCI), and incubated at 37°C. Table 3 shows that neither incubation time, nor the solvent pH made any appreciable difference to the solubility of chromium. The total chromium recovery varied within a narrow range of 92104% in the worst case with respect to incubation time. The results were further confirmed by repeating the experiment five times at an incubation time of 16 h (see Table 4). The RSDs between trials were 1.7% and 2.7% under neutral and acidified conditions, respectively. Thus, other samples were analyzed after a oneday period of standing time at room temperature unless otherwise noted.

3.2. Ion-exchange chromatography The chromatography of speciation of Cr(IIl) and Cr(VI) had been developed earlier in our laboratory [20] and was employed here to investigate the presence of ionic chromium species, especially Cr(VI), in the chromium picolinate samples. Detailed chromatographic conditions can be found in Ref. [20] and are briefly illustrated in Table 5. The free cationic Cr(III) needed to be chelated with EDTA for good

1806

H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

CrP tablet (50ag) 40 35

8

30

25 15

5 0

1

2

3

4

5

Time (min)

CrP synthesized (20ng) 14 12 10

6

Ill'v" 1" ~'~

~

-

' " ~

~ "r=

"q~w

I

V

Time (rain) Fig. 5. Separationof chromiumpicolinate samples using RPLC. C-18 column, 60% MeOH,flow rate 1 ml rain-~. Injectionvolume20/~1. (a) Filtrate of commercialproduct,2.5 ppm (as Cr). (b) Filtrateof synthesizedproduct, 1 ppm (as Cr). reproducibility (in terms of retention time and peak height). This complication was presumed to be caused by the slightly cationic properties of the column [20]. Fig. 1 shows an injection of a standard mixture of Cr(III)-EDTA and Cr(VI) using ICP-MS detection. Fig. 2 is a chromatogram of an injection of chromium picolinate filtrate. It is clear that there was no detectable amount of Cr(VI) present. The two chromium-containing species present did not match the standard retention times (tR). Figs. 3a and 3b are chromatograms of chromium picolinate samples spiked with 100 ppb Cr(III)EDTA and Cr(VI), respectively, incubated for 2 h. In order to study the possible changes that the chromium

picolinate species may undergo when subjected to stomach conditions, chromium picolinate samples were adjusted to pH 3.7 with HCI (the value for gastric juices is pH 3-4 [22]), and incubated at 37°C for different periods of time. It was found that incubation under acidic conditions for less than 4 h did not facilitate conversion of the chromium species to other forms. 3.2.1. Synthesis o f chromium picolinate

Synthesis of chromium picolinate was attempted following a simple procedure [23]. 0.01 mol chromium chloride hexahydrate was dissolved in 25 ml deionized, distilled water, followed by the

H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

20

~

mm ~

J

~

J

~

1807

m

it

15

|11° 5 0

10

2

~

3

4

5

Time (rain)

1o 9 8

7 5 4 3

k__ T i m e (min)

Fig. 6. Injection of commercial chromium picolinate filtrates using RPLC. LC conditions are the same as in Fig. 5. (a) Sample concentration 10 ppm. (b) Sample concentration 5 ppm.

addition of 0.03 mol picolinic acid. The solution was stirred at room temperature until a reddish complex was precipitated. This precipitate was filtered, washed with deionized water, and air dried.

3.2.2. Analysis of synthesized chromium picolinate by ion-exchange chromatography The synthesized chromium picolinate was subjected to chromatographic analysis. Fig. 4 shows that two forms of chromium, with similar tRS to those of the commercial chromium picolinate tablets, were observed. This indicates that the synthesized and commercial chromium picolinate are consistent in terms of chromium species. Since the precise identification of the two chromium species was unclear, Cr(III)-EDTA was used as a standard for their quantification. This method pro-

vides an estimate of Cr concentration since each compound has a unique concentration/peak-count relationship. Peak 1 was estimated to have about 30 ppb chromium and 60 ppb chromium for the second peak (Fig. 2), giving a total concentration of 90 ppb elemental chromium. Chelating the chromium picolinate gave similar recoveries for both species (30 ppb and 70 ppb, respectively); thus, the total chromium content determined by LC-ICP-MS was only around 100 ppb. This provides only 1% recovery compared to the 9 ppm total chromium obtained by direct nebulization analysis. Apparently, most of the chromium was lost in the chromatography. We believe that the poor recovery can be attributed to the retention of chromium species on the column(s). The IonPac AS7 is a hydrophobic anion-exchange column, which may react with the free Cr(III) cations.

1808

H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

20 15

1 10

°o

i

!

!

4

5

Time (rain)

15

1! 1°

00 Time (rain) Fig. 7. Injection of commercial chromium picolinate filtrates using RPLC. LC conditions are the same as in Fig. 5. (a) Sample concentration 40 ppm. (b) Sample concentration 20 ppm.

It requires an NG1 guard column (reversed-phase) to remove the non-ionic organic compounds from the sample which may appear to complicate the analysis of chromium ions [24]. Apparently, the organic form(s) of chromium picolinate may be retained by the two columns. We found that the background signals increased by a factor of 10 after 30-40 injections of chromium samples, and the guard column contributed significantly to the increase. It became clear that other types of chromatography, especially reversedphase chromatography, would be necessary for further studies.

3.3. Reversed-phase chromatography The mobile phase methanol concentration was varied to find the best peak shape and signal-to-back-

ground ratio. This part of the preliminary study was carried out using the Dionex HPLC system with UV detection at 222 nm. Table 6 indicates that 60% methanol yielded the best chromatographic results, giving the optimum compromise between signal-tobackground ratio, capacity factor, and peak symmetry (a symmetry factor of 1 represents a gaussian peak). Table 6 Effect of MeOH concentration on chromatographic characteristics, capacity factor (k'), peak symmetry factor (B/A), and signal-tobackground ratio (S/B) MeOH

30%

60%

80%

100%

k'

1.85 1.15 1600

0.78 1.2 2200

0.68 1.4 1570

0.94 Split peaks 550, 300

B/A S/B

H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

14 12 10 6

I,"r "~lw'"~ - ' " r ' . . . . -~' ,r~,rlnV,ig~

2 0

o

a

1o

8

4

;

;

.

1o

4

2 o

¢. h n ~ 4

i

o

1'o

Time (min) Fig. 8. Analysis of commercial chromium picolinate products, using ion-exchange chromatography. LC conditions are the same as in Fig. 1. All samples were filtrates prepared by dissolving one tablet of different brand products in 20 ml water.

ICP-MS detection was later used for further investigation. The ICP operating conditions were the same as listed in Table 1. Fig. 5a is a chromatogram for the injection of the commercial chromium picolinate tablet sample using this reversed-phase liquid chromatography (RPLC) method. Compared to Fig. 2, which shows the results from ion-exchange chromatography, the chromium recovery by RPLC was much higher. However, it was difficult to identify whether the chromium species

1809

detected by RPLC was the same as either of the two forms found by ion-exchange chromatography. By following the same estimation scheme, we found that the recovery of chromium by RPLC was nearly complete. It is assumed that the species found by RPLC gave the major chromium picolinate peak. Table 7 shows a comparison of chromium concentrations presented by this RPLC-ICP study with the labeled values. As observed, the RPLC-ICP data appeared high, giving more than 100% recovery. This overestimation was possibly due to the use of Cr(III)-EDTA as the reference. Nevertheless, full recovery was achieved. This suggested that the low chromium recoveries associated with ion-exchange chromatography occurred because the majority of the chromium was not ionic. This major peak is believed to be the chromium picolinate form. No background increase after repeated sample injection was observed. Fig. 5b shows that the chromatogram of synthesized chromium picolinate compares favorably with that of the commercial chromium picolinate. The recoveries at concentrations from 100 ppb to 10 ppm for synthesized chromium picolinate by RPLC-ICP compared favorably with the expected concentrations (Table 8). In Fig. 5b, a small peak appeared before the major chromium species. This was also observed with the concentrated chromium picolinate tablet sample. Figs 6 and 7 show injections of four chromium picolinate samples of increasing concentration (peaks are offscale to show details). These small peaks may be the binuclear chromium picolinate complexes, isomers of the mono- or binuclear chromium picolinate, or even their decomposed forms. According to Steam and Armstrong [25], two products, the reddish mononuclear Cr(pic)3 and the purple binuclear [Cr(pic)2OH] 2, can be formed from the aqueous reaction mixtures of Cr(III) and picolinic acid. The dimer does not crystallize readily, forming slowly after a few days. Although the synthesis in this experiment was carried out in 4 h, the possibility of dimer formation could not be ruled out.

3.4. Other chromium picolinate-containing products Three other chromium picolinate-containing commercial products have also been studied with RPLC.

1810

H. Ding et al./Spectrochiraica Acta Part B 51 (1996) 1801-1812 100 90 80

a. Immd2

70

1

30

A_...._

10 O0

1

2

30 /

3

4

||1

ii

b. bnmd3 [

25 ]'

10

ill.,.

m

•

~0

m

m

i

1

2

3

4

5

1

2

3

4

$

Time (rain)

Fig. 9. Analysis of commercial chromium picolinate products, using reversed-phasechromatography.LC conditions are the same as in Fig. 5. All samples were filtrates prepared by dissolving one tablet of different brand products in 20 ml water.

Table 7 Chromium concentration determined by RPLC-ICP-MS vs. the commercially available value labeled on the tablet bottle Cr recovery by RPLC-ICP-MS/% 1 tablet/320 ml 1 tablet/160 ml 1 tablet/80 ml l tablet/40 ml 1 tablet/20 ml

110 104 108 108 109

% RSDa

2.1 2.0 2.4 4.1

Figs. 8 and 9 represent their analysis by ion-exchange and RPLC, respectively. As before, the chromium recovered by ion-exchange chromatography was poor. Only small peaks were observed as chromiumcontaining species, while the more predominant peaks were due to the presence of C1- from the matrix (CIcan form C10 + in the plasma which also appears at m/z 53). However, RPLC gave more quantitative results (Fig. 10). Even though only peak 1 in Fig. 9a matched the retention time of chromium picolinate in Fig. 5, it is possible that different commercial

H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

1811

120 II0 I00 r

90 8O

7O 60

'

I

I

I

I

I

CrPIl60ml(l.3ppm) CrP/40ml(5ppm) CrP/lOml(2Oppm) CrP/320ml(0.63ppm) CrPIS0ml(2.Sppm) CrP/20ml(10ppm) CrP/Sml(40ppm)

. , . ICP data

Solvent (ml) - - - LC-ICP data

Fig. 10. Comparison of chromium recovery by direct solution nebulization and LC sample introduction.

products vary in the type and amount of chromium picolinate; chromium picolinate itself can exist in the form of monomers and dimers.

Fig. 5a was believed to be due to chromium picolinate. Other minor peaks may be attributable to other chromium picolinate-containing complexes, such as chromium picolinate dimer or chromium dinicotinate, and some decomposition products.

4. Summary Our study shows that several of the chromium picolinate products currently on the market do not contain elemental Cr(VI). Ion-exchange chromatography used in this work had a tendency to retain the organic chromium picolinate. RPLC proved to be a more effective approach. As evident in Fig. 10, a representative comparison of the chromium recovery obtained by the ICP and RPLC-ICP study, chromium recovery proved to be complete with RPLC. The major peak observed in Table 8 Cr recovery of synthesized chromium picolinate by different methods of determination Expected concentration/ppm

Direct Neb.-ICPMS/%

RPLC-ICP-MS/%

100 1 10

97 97 91

107 98 92

Acknowledgements The authors are grateful to the National Institute of Environmental Health Sciences for support of this work through grant No. E504908.

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H. Ding et al./Spectrochimica Acta Part B 51 (1996) 1801-1812

(Ed.), Quick Reference to Clinical Nutrition: A Guide for Physicians, JB Lippincott, Philadephia, PA, 1987. [8] V.W. Bunker, M.S. Lawson, H.T. Delves and B.E. Clayton, Am. J. Clin. Nutr., 39 (1984) 799. [9] A.S. Kozlovsky, P.B. Moser, S. Reiser and R.A. Anderson, Metabolism, 35 (1986) 515. [10] R.A. Anderson, M.M. Polansky, N.A. Bryden, K.Y. Patterson, C. Veillon and W. Glinsmann, J. Nutr., 113 (1983) 276. [11] J.S. Borel, T.C. Majerus, M.M. Polansky, R.B. Moser and R.A. Anderson, Biol. Trace Elem. Res., 6 (1984) 317. [12] R.A. Anderson, M.M. Polansky, N.A. Bryden, E.E. Roginski, K.Y. Patterson, C. Veillon and W. Glinsmann, Am. J. Clin. Nutr., 36 (1982) 1184. [13] W. Mertz, Physiol. Rev., 49 (1969) 163. [14] R. Anderson, Clin. Physiol. Biochem., 4 (1986) 31. [15] Committee on Dietary Allowances, Food and Nutrition Board, National Reserach Council: Recommended Dietary Allowances, 9th revised edn., National Academy of Sciences, Washington, DC, 1980.

[16] M.F. Mccarty, Med. Hypotheses, 41 (1993) 316. [17] G.W. Evans and D.J. Pouchnik, J. Inorg. Biochem., 49 (1993) 177. [18] J. Huszonek, Am. J. Psychiatr., 150 (1993) 1560. [19] B. Buckely, W. Fang, W. Johnson and C. Gilmartin, Is there Cr(VI) in the mineral suppliments you are taking?, presented at the FACSS XXII Conference, Rutgers University, Cincinnati, OH, USA, October 15-20, 1995. [20] F.A. Byrdy, L.K. Olson, N.P. Vela and J.A. Caruso, J. Chromatogr., 712 (1995) 311. [21] Y. Suzuki and F. Serita, Ind. Health, 23 (1985) 207. [22] J.J. Alexander and M.J. Steffel, Chemistry in the Laboratory, 2nd edn., Edina, MN, Burgess Int. Group, 1988, p. 213. [23] G.W. Evans and T.D. Bowman, J. lnorg. Biochem., 46 (1992) 243. [24] Technical Note 26, Dionex Corp., Sunnyvale, CA, May 1990. [25] D.M. Steam and W.H. Armstrong, Inorg. Chem., 31 (1992) 5178.