ANALYTICAL SCIENCES SEPTEMBER 2004, VOL. 20 1321 2004 © The Japan Society for

Estimation of Trace Amounts of Chromium(III) in Multi-Vitamin with Multi-Mineral Formulations

Jolly M. SANKALIA, Rajashree C. MASHRU,† Mayur G. SANKALIA, Priti P. PARIKH, and Vijay B. SUTARIYA

Pharmacy Department, Faculty of Technology & Engineering, The Maharaja Sayajirao University of Baroda, Kalabhavan, Vadodara, Gujarat 390001, India

Two new specific, selective, simple and inexpensive spectroscopic methods for estimating a trace amount of chromium (Cr3+) from a multi-vitamin with multi-mineral pharmaceutical formulations were developed. The proposed methods are based on the conversion of Cr3+ to Cr6+ either by oxidation with a nitric acid–perchloric acid mixture (method I) or by fusion with an excess amount of sodium carbonate (method II), followed by the complexation of Cr6+ with 1,5- diphenylcarbazide (DPC) in a mineral acidic solution of pH 1.0 ± 0.5. The pink-colored complex was estimated at 544 nm. Both methods were found to be linear in the range of 0.1 – 0.8 µg/ml with a limit of detection in the range of 0.0123 – 0.0157 µg/ml and a limit of quantitation in the range of 0.0419 – 0.0525 µg/ml. Method I was found to be suitable for estimating Cr3+ species in various formulations, like tablets, capsules and syrups, while method II was found to be suitable for tablets and capsules. Satisfactory recovery from spiked samples of standard Cr3+ suggests no interference of any excipients and diverse ions present in the formulations. The developed methods were compared with AAS by ANOVA, and no significant difference was observed.

(Received February 4, 2004; Accepted May 28, 2004)

tea and tea leaves, food, dietary products, lung tissues, standard Introduction alloys and biological samples; zeeman AAS from blood and serum; electro thermal AAS (ETAAS) from cocaine samples; Chromium (Cr) can exist in various chemical valence states AAS–flame emission (AAS-FES) from ranging from Cr2+ to Cr6+, among which more stable chemical ancient Perivian hair; mass (MS) from metal forms are Cr3+ and Cr6+. Cr3+ is relatively non-toxic and is an allergenic substances on skin; inductively coupled plasma–mass essential nutrient in the human diet to maintain effective spectrometry (ICP-MS) from seawater; ICP-atomic emission glucose, lipid and protein metabolism.1 However, Cr6+ is spectroscopy (ICP-AES) from human gallstone; neutron 2– 4– primarily anthropogenic and, as CrO4 or HCrO , can diffuse activation analysis (NAA) from biological specimens, tissue through cell membranes and oxidize biological molecules with surroundings, blood and milk; electron spin resonance (ESR) toxic results.2 Cr6+ has been classified by the International from rat blood; scanning electron microscopy (SEM), Agency for Research on Cancer (IARC) as a Group I human transmission electron microscopy (TEM) and electron probe carcinogen3 and by the US Environmental Protection Agency microanalyses (EPMA) from metal-bearing contaminated soil (EPA) as a Group A inhalation carcinogen.4 and industrial waste; colorimetric methods6 from shrimp feed,7 Chromium is one of the essential trace elements in multi- air,8,9 urine8,10,11 and catgut suture,12 etc. vitamin with multi-mineral pharmaceutical formulations that Not even a single spectroscopic method has been reported for contain only Cr3+ either in the form of chromium chloride estimating Cr3+ from pharmaceutical formulations. Thus the (inorganic source) or chromium picolinate and chromium primary objective of the present work was to develop, optimize polynicotinate (organic source). The methods for estimating and validate sensitive, selective, precise, accurate, cost- Cr3+ from pharmaceutical formulations are atomic absorption effective, and simple spectroscopic methods. spectroscopy (AAS) and graphite furnace AAS (GF-AAS). The proposed methods are based on removing all organic Other methods reported in the literature for estimating Cr3+ are matter of a multi-vitamin with multi-mineral pharmaceutical chemiluminescence analysis, flame AAS, X-ray fluorescence formulations by heating in a muffle furnace13 at 550˚C – 600˚C spectroscopy (XRFS) and colorimetric from air.5 Various for 6 – 7 h. Cr3+ was converted to Cr6+ by oxidation with a nitric methods for estimating Cr6+ and total Cr include flow-injection acid–perchloric acid mixture13 (method I) and by fusion with flame AAS from industrial effluents; turbidimetry; ion excess of sodium carbonate14 (method II), followed by ; anion-exchange chromatography; HPLC with estimating a pink-colored complex in a mineral acidic solution diode array detection (HPLC-DAD) from environmental of pH 1.0 ± 0.5 at 544 nm obtained by the reaction of Cr6+ with matrices; fast protein liquid chromatography (FPLC); thermal 1,5-diphenylcarbazide (DPC), which is considered to be one of lens spectrometry (TLS); X-ray fluorescence; AAS from black the most sensitive and selective reactions for Cr6+ determination.15–19 The developed methods were studied † To whom correspondence should be addressed. concerning the effect of the pH, dye volume, Cr6+:dye ratio, E-mail: [email protected] stability of the colored complex, etc. for methods I and II, while 1322 ANALYTICAL SCIENCES SEPTEMBER 2004, VOL. 20

Fig. 1 Visible spectra of Cr6+: dye complex at 0.1 µg/ml (a), 0.2 µg/ml (b), 0.3 µg/ml (c), 0.4 µg/ml (d), 0.5 µg/ml (e), 0.6 µg/ml (f), 0.7 µg/ml (g) and 0.8 µg/ml (h) concentration levels.

Fig. 2 Optimization of the Na2CO3 composition using chromium the Na2CO3 composition, heating time were considered for method II. chloride as a standard.

Experimental 0.5 and the volume was made to obtain 5 µg/ml of Cr6+. From this stock solution, suitable aliquots were taken and 0.2 ml of Apparatus DPC dye was added to each, allowed to stand for 5 min for full A Shimadzu UV-VIS spectrophotometer (Model UV-1601) color development and the volume was made up to 10 ml with with matched 1 cm silica cells was used for all spectral and distilled water to obtain the final concentration in the range of absorbance measurements. The glassware used in each 0.1 to 0.8 µg/ml; the absorbance was measured at 544 nm. procedure was soaked overnight in a chromic mixture (K2Cr2O7 + concentrated H2SO4), rinsed thoroughly with double-distilled Preparation of a sample solution (method I) water and dried in dust-free air. Whatman filter paper No. 42 After twenty tablets/capsules were weighed and triturated, a was used to filter solutions of different formulations to separate powder equivalent to 1.0 mg of Cr3+ was subjected to heating in them from the solvent immiscible formulation excipients. a muffle furnace as per the conditions stated under “Preparation of standard solution for (method I)”. The ash Reagents was then digested with 30 ml of a nitric acid–water–perchloric Double-distilled water and analytical-reagent grade chemicals acid mixture (5:5:20 v/v/v) on a burner until white fumes of were used. Standard solutions of chromium were prepared by perchloric acid started to appear, became vigorous and then using chromium chloride hexahydrate (CrCl3·6H2O) or ceased. This solution was allowed to cool, and was diluted with 6+ chromium picolinate (C18H12N3O6Cr) salts. A Cr complexing distilled water; the pH was adjusted at 1 ± 0.5 and the volume reagent solution of 1,5-diphenylcarbazide (DPC) (S. D. Fine was made to obtain a final concentration of 5 µg/ml of Cr6+ to Chem Ltd., Mumbai, India) was prepared by dissolving 0.25 g proceed further, as mentioned above. of DPC in 50 ml of acetone, and stored in an amber-colored bottle. This solution was discarded when it became slightly Preparation of a sample solution (method II) colored, and should not be used after 48 h of its preparation; it is After twenty tablets/capsules were weighed and triturated, a always preferable to use it freshly prepared. powder equivalent to 1.0 mg of Cr3+ was mixed with sodium

carbonate (Na2CO3) equivalent to 5-times the weight of the Preparation of standard solution for calibration curve (method I) sample taken, mixed/triturated well and then subjected to Chromium salts (chromium chloride hexahydrate or heating in a muffle furnace, as described under “Preparation of chromium picolinate) equivalent to 1.0 mg of Cr3+ were standard solution for calibration curve (method II)”. The ash subjected to heating in a muffle furnace in a silica crucible at was dissolved in about 100 ml of water and boiled on a water- about 550˚C for 6 – 7 h; the ash was then dissolved in 0.2 M bath for about 45 min, cooled and filtered. The pH of the sulfuric acid (pH 1 ± 0.5) in order to obtain a stock solution of 5 filtrate was adjusted at around 1 ± 0.5, and the volume was µg/ml of Cr6+. From this stock solution, suitable aliquots were made to obtain 5 µg/ml of Cr6+ to proceed further, as mentioned taken and 0.2 ml of DPC dye was added to each, allowed to above. stand for 5 min for full color development and volume was made up to 10 ml with distilled water to obtain the final concentration in the range of 0.1 to 0.8 µg/ml; the absorbance Results and Discussion was measured at 544 nm. Absorption spectra Preparation of standard solution for calibration curve (method II) The absorption spectra of a Cr6+-DPC dye complex are In method II, chromium salts (chromium chloride hexahydrate graphically presented in Fig. 1. As can be seen, the complex or chromium picolinate) equivalent to 1.0 mg of Cr3+ along with has its maximum absorbance at 544 nm. sodium carbonate (Na2CO3) equivalent to 5-times its weight were heated in a muffle furnace in a silica crucible at about Effect of the Na2CO3 composition (method II) 550˚C for 6 – 7 h, and then the ash was dissolved in about 100 The Na2CO3 composition in method II is a very critical ml of water, boiled on a water-bath for about 45 min, cooled parameter, which was investigated for optimum conditions. and filtered. The pH of the filtrate was adjusted at around 1 ± Na2CO3 equivalent to 4-times, 5-times and 6-times of the ANALYTICAL SCIENCES SEPTEMBER 2004, VOL. 20 1323

Fig. 5 Effect of the dye volume using chromium chloride as a standard. a, Method I; A, Method II.

Fig. 3 Optimization of the heating time using chromium chloride as a standard.

Fig. 6 Job’s plot using chromium chloride as a standard. a, Method I; A, Method II.

Fig. 4 Effect of the pH using chromium chloride as a standard. Cr6+:dye complex was drastically reduced as the pH increased, a A , Method I; , Method II. and because it was almost constant over the range of 1 ± 0.5, it was selected as being optimum. The results for chromium chloride salt for both methods are shown in Fig. 4. sample weight was taken for both of the chromium salts (i.e. chromium chloride or chromium picolinate). The same process Effect of the dye volume (methods I and II) was applied to all of the samples, and the absorbance at 544 nm For evaluating the effect of the dye volume, different aliquots was measured. Na2CO3 equivalent to 5-times the sample weight (0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.8, 1, and 1.5) of was found to be the optimum composition for the complete 0.5% w/v DPC in acetone were taken for the same oxidation of Cr3+ to Cr6+. Below this level, oxidation was concentration of Cr6+ (0.7 µg/ml). A dye volume of 0.2 ml of incomplete and above this level it was interfered. This was 0.5% w/v was found to be optimum for both salts, because at further correlated from the results shown in Fig. 2, where a less volume the dye quantity was insufficient to form a Cr6+:dye Na2CO3 equivalent to 5-times the sample weight showed the complex, and at higher volumes of the dye, the complex maximum absorbance for both salts. formation was interfered. As presented in Fig. 5, it showed the maximum absorbance at 0.2 ml of dye volume for chromium Effect of the heating time (method II) chloride salt for both methods. Because boiling the ashed samples with 100 ml of water in a water-bath is required for complete conversion of Cr3+ to Cr6+ in Chromium (Cr6+):dye ratio (Job’s plot) method II, the effect of the boiling time was studied with In order to determine the Cr6+:dye ratio, equimolar (0.0004 M) different time intervals of 15, 30, 45 and 60 min. As shown in solutions of chromium (Cr6+, chromium chloride used as a Fig. 3, boiling for less than 45 min was insufficient for complete standard salt) and DPC dye were prepared and different mole conversion, and boiling for more than 45 min yielded in less % fractions of chromium and dye were taken. The absorbance was label claim. Hence, a boiling time of 45 min was found to be plotted against mole fraction of DPC to determine the ratio. optimum, because it showed the maximum % label claim for This plot is called Job’s plot. As shown in Fig. 6, Cr6+:dye ratio both salts. of 3:7 showed the maximum absorbance, indicating a 1:2 complex for both methods. Effect of the pH (methods I and II) For the stability of the oxidized Cr6+ species, an acidic Stability of colored complex environment is essential. Organic compounds and alkaline No significant change was observed in the absorbance for up condition can cause Cr6+ reduction to Cr3+, which will not form to 3 h, but a significant reduction in absorbance during the forth a complex with DPC dye. This was confirmed by maintaining hour indicated the stability of the colored complex to be of 3 h. different pH values (0.1, 0.33, 0.5, 0.75, 1, 1.2, 1.4, 1.6, 1.8, 2, 3, 5, and 6.8) of a solution containing the same concentration of Calibration curve, sensitivity and validation oxidized Cr6+ species (0.7 µg/ml). The color intensity of the Under the recommended conditions, the system obeys Beer’s 1324 ANALYTICAL SCIENCES SEPTEMBER 2004, VOL. 20

Table 1 Optical characteristics and analytical data

Method I Method II Parameter Chromium chloride Chromium picolinate Chromium chloride Chromium picolinate

Absorption maxima, λmax/nm 544 544 544 544 Linearity range/µg ml–1 0.1 – 0.8 0.1 – 0.8 0.1 – 0.8 0.1 – 0.8 Molar extinction, ε /l mol–1 cm–1 1.7755 × 104 1.8668 × 104 1.7604 × 104 1.8703 × 104 Sandell’s sensitivity/µg cm–2 2.9285 × 103 2.7852 × 103 2.9536 × 103 2.7801 × 103 Coefficient of determination (r2) 0.9996 0.9992 0.9997 0.9991 Correlation coefficient (r) 0.9998 0.9996 0.9998 0.9995 Regression equation (Ya) Slope (b) 0.3028 0.3087 0.3049 0.3097 Intercept (a) 0.0116 0.0151 0.0101 0.0150 Limit of detection, LoD/µg ml–1 0.0126 0.0157 0.0123 0.0128 Limit of quantitation, LoQ/µg ml–1 0.0419 0.0525 0.0409 0.0425 a. Y = a + bx, where x is the concentration (µg/ml). LoD = 3σσσ /S, LoQ = 10 /S, where is standard deviation of blank, and S is slope of calibration.

Table 2 Inter-day (n = 3) and intra-day (n = 6) precision and Table 3 Recovery study of pharmaceutical formulations accuracy Standard Labeled Labeled Cr Cr Labeled Formu- Cr Chromium Recovery, Formu- Cr Method added/ RSD Method founda/ claim, SD RSD lation claim/ founda/µg % lation claim/ µg µg % µg µg (CrCl3) Inter-day I Tablet-1 65 63.78 98.12 1.12 1.14 13 12.92 99.39 1.51 Tablet-1 65 (n = 3) Capsule-1 65 64.43 99.13 1.97 1.99 26 25.63 98.58 1.71 Syrup-1 25 25.22 100.87 1.45 1.44 13 13.04 100.35 1.50 I Capsule-1 65 II Tablet-1 65 64.19 98.77 1.40 1.42 26 26.09 100.38 1.68 Capsule-1 65 64.44 99.13 1.61 1.63 5 5.05 101.09 2.04 Syrup-1 25 Intra-day I Tablet-1 65 64.49 99.22 1.27 1.28 10 9.94 99.39 1.59 (n = 6) Capsule-1 65 64.55 99.31 1.42 1.42 13 13.13 100.99 1.48 Syrup-1 25 25.26 101.06 1.74 1.72 Tablet-1 65 26 26.08 100.30 1.09 II Tablet-1 65 63.72 98.04 1.18 1.20 II 13 12.88 99.08 1.50 Capsule-1 65 64.44 99.13 1.57 1.58 Capsule-1 65 26 25.99 99.99 1.09 a. Average of three and six determinations at two concentration level a. Average of three determinations. for inter-day and intra-day, respectively.

carried in the presence of interfering ions in order to judge law over the concentration range of 0.1 – 0.8 µg/ml for both whether the estimation is interfered or not. Good recovery for salts and for both methods with an RSD of less than 1% at each the added salt, as shown in Table 4, indicated no interference of concentration level. The low values of the standard error and the diverse ions on the estimation of chromium. the coefficient of variation establish the precision of the proposed methods. Other parameters, like the molar Application absorptivity, Sandell’s sensitivity, regression equation, The developed methods can be successfully applied for coefficient of determination (r2), correlation coefficient (r), limit estimating Cr3+ from various pharmaceutical multi-vitamins of detection (LoD), limit of quantitation (LoQ), etc. are given in with multi-mineral formulations, such as tablets, capsules and Table 1. The obtained correlation coefficient values are highly syrups. However, method II is applicable for estimating Cr3+ significant for both methods. Inter-day and intra-day RSD were from tablets and capsules. The analyses of various also found to ascertain the precision of the developed methods. pharmaceutical formulations are enumerated in Table 5. As shown in Table 2, RSD did not exceed 2% in any case. Recovery studies were carried out to ascertain the accuracy and Comparison with AAS precision of the methods by spiking the samples with known The results of method I, method II and AAS13 were compared amounts of standard chromium salt to previously analyzed the statistically by ANOVA. The reported F value, as given in samples. Good recoveries of the analytes were attained (Table Table 6, of a two-way ANOVA test for difference between 3) with RSD less than 2.0%. methods, suggests that there is no significant difference.

Effect of diverse ions on the determination of chromium From the discussion above, it is clear that the developed For studying the effect of diverse ions, a homogenous mixture methods are accurate, precise, repeatable, reproducible, linear, containing various ions (like zinc, manganese, copper, iron, quicker, inexpensive, sensitive and simple. Therefore, the silicon, selenium, iodide, molybdenum, vanadium, etc.) in proposed methods make them useful in the routine and quality different forms was prepared containing standard chromium salt control analysis of multi-vitamins with multi-mineral (chromium chloride) in a known quantity; the estimation was pharmaceutical formulations. Moreover, no significant ANALYTICAL SCIENCES SEPTEMBER 2004, VOL. 20 1325

Table 4 Effect of diverse ions on the estimation of chromium Table 6 Two-way ANOVA test for determining the difference between the methods Ion Added as Quantity of ion Claim by Claim by Claim by 2+ Zn ZnSO4 15 mg Formulation method I, method II, AAS13, Total 2+ Mn MnSO4 2 mg % % % 2+ Cu CuSO4 2 mg 2+ Fe FeSO4 15 mg Tablet-1 99.31 98.58 100.23 298.13 4+ Si SiO2 2 mg Tablet-2 100.78 100.41 104.36 305.55 4+ Se Na2SeO4 150 µg Capsule-1 99.77 98.95 96.98 295.70 I– KI 150 µg Capsule-2 99.50 98.13 99.66 297.28 6+ Mo (NH4)6Mo7O24 75 µg 2+ Source of variation DF SS MS Fcal Ftab V Na4VO3 10 µg 3+ µ Cr CrCl3 200 g Between the methods 2 3.43 1.71 0.79 5.14a 3+ µ Cr found 196.74 g Between the formulations 3 19.12 6.37 2.94 4.76b Recovery, % 98.37% Residual 6 13.00 2.17

DF, degree of freedom; SS, sum of squares; MS, mean sum of squares. Table 5 Results of the analysis of pharmaceutical a. Theoretical value of F(2,6) based on two-way test at p = 0.05 level formulations of significance. Using chromium Using chromium b. Theoretical value of F(3,6) based on two-way test at p = 0.05 level chloride picolinate of significance. Labeled Formu- calibration curve calibration curve claim/ lation µg Amount Labeled Amount Labeled foundc/ claim, foundc/ claim, µg % µg % Research on Cancer, Lyon. 4. USEPA 600/8-83-014F, “Health Assesment Document for Method I Chromium”, 1984, US Environmental Protection Agency, a Tablet-1 65 64.55 99.31 —— Washington, D.C. Tablet-2b 200 ——203.19 101.60 5. T. S. Burenko, L. P. Artem’eva, and G. D. Vlasova, Gig. Capsule-1a 65 64.85 99.77 —— Capsule-2b 150 ——147.27 98.18 Tr. Prof. Zabol., 1989, 9, 46. Syrup-1a 25 25.36 101.42 —— 6. N. Hara, Ind. Health, 1986, 24, 41. Method II 7. S. Divakaran, L. G. Obaldo, and I. P. Forster, J. Agric. Tablet-1a 65 64.08 98.58 —— Food Chem., 2002, 50, 464. Tablet-2b 200 ——201.81 100.90 8. T. Dutkiewicz, J. Konczalik, and M. Przechera, Acta Pol. Capsule-1a 65 64.32 98.95 —— Pharm., 1969, 26, 165. Capsule-2b 150 ——148.13 98.75 9. W. Matczak, T. Rogaczewska, and J. T. Solnica, Med. Pr., 1986, 37, 197. a. Formulations containing Cr3+ in the form of chromium chloride. 10. Y. Q. Li, Y. H. Zhao, Y. H. Cao, and M. Z. Shao, Hua. Xi b. Formulations containing Cr3+ in the form of chromium picolinate. c. Average of three determinations at two concentration level. Yi Ke Da Xue Xue Bao., 1989, 20, 85. 11. G. H. Zhu, Zho. Yu Fang Yi Xue Za Zhi, 1984, 18, 173. 12. W. J. Smith, M. A. Johnston, and A. R. Lea, J. Pharm. Pharmacol., 1984, 36, 687. difference was observed between the developed methods and 13. “United States Pharmacopeia, USP-25/NF-20”, 2002, the pharmacopoeial method (AAS). Authority of the United States Pharmacopeial Convention, Washington, D.C., 2460. 14. E. B. Sandell, “Colorimetric Determination of Traces of Acknowledgements Metals”, 1959, Intersciences, New York, 388. 15. A. D. Eaton, L. S. Clesceri, and A. E. Greenberg, The authors are thankful to the management and workers of the “Standard Methods for the Examination of Water and Relax Pharmaceuticals, Vadodara, India and Elysium Water Waste”, 1985, American Public Health Association, Pharmaceuticals, Vadodara, India, for participating in this study American Water Works Association, Water Pollution by providing laboratory technical assistance. Control Federation, Washington D.C., 201. 16. USEPA Method 7196A, “Test Methods for Evaluating Solid Waste”, 1990, US Environmental Protection Agency, References US Government Printing Office, Washington, D.C. 17. NIOSH, “NIOSH Manual of Analytical Methods”, 1994, 1. S. A. Ketz and H. Salem, “The Biological and National Institute for Occupational Safety and Health, Environmental Chemistry of Chromium”, 1994, VCH, New Cincinnati. York, 43. 18. USEPA Method 3060A, “Alkaline Digestion for 2. M. Stoeppler, “Hazardous Metals in the Environment: Hexavalent Chromium”, 1996, US Environmental Techniques and Instrumentation in Analytical Chemistry”, Protection Agency, US Government Printing Office, 1992, Elsevier, Amesterdam, 373. Washington, D.C. 3. IARC, “IARC Monographs of the Evaluation of the 19. OSHA, “Hexavalent Chromium in Workplace Carcinogenic Risk of Chemicals to Humans: Chromium, Atmosphere”, 1998, Occupational Safety and Health Nickel and Welding”, 1990, International Agency for Association, US Department of Labor, Washington, D.C.