Research Journal of Chemical Sciences ______ ______________________________ ______ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 46 - 51 , March (201 2 ) Res.J.Chem.Sci. International Science Congress Association 46 AAS Estimation of Heavy Metals and Trace elements in Indian Herbal Cosmetic Preparations Kumar Sukender, Singh Jaspreet , Das Sneha and Garg Munish * Department of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak 124001, INDIA Available online at: www.isca.in (Received 21 st January 2012 , revised 27 th January 2012 , accepted 9 th Februry 2012 ) Abstract In present study, heavy metals like Pb, Cd and trace (micronutrients) metals like Ca, Mg, Al, Cu and Zn were quantitatively estimated using flame atomic absorption spectrometry (FAAS). Also, heavy metals such as As and Hg were estimated by hydride generati on technique (cold vapour atomic absorption spectrometry) using nitrogen as carrier gas in 21 herbal cosmetic preparations sold in Indian market. The results indicate that among the toxic heavy metals, two samples for Hg content and si x for Pb content were exceeding the WHO permissible limits fixed for herbal preparations. Arsenic was found appreciably well below the permissible limit, but Cd was found above the permissible limit in the all samples. Trace elements like Ca and Mg were found in higher amount than Al, Cu, and Zn. Presence of trace elements can prove to be beneficial but presence of toxic heavy metals in such amounts surely has adverse effects on the consumer health who always take the herbal products in an impression of being safe because of t he natural origin. In conclusion, enforcement of strict and separate regulatory guidelines and promotion of Good analytical practice (GAP), good manufacturing practices (GMP) and good agricultural and control practices (GACP) is suggested for herbal cosmet ics by WHO and other regulatory agencies. This study presents the status of heavy metals and trace elements in marketed herbal cosmetic formulations and also provides a simple and convenient AAS method which can effectively be adopted at Industrial level f or the quality control and standardization of herbal cosmetic preparations and other related products. Keywords: Herbal cosmetics, trace elements, heavy metals, AAS . Introduction Herbal cosmetics are the valuable products consist of botanicals or their bioactive ingredients/extracts which enrich the skin with trace (nutrient) elements and other useful minerals, prevent from infection and hence responsible for their cosmetic effects. Although they are used worldwide since ancient time, but in the last decade, ther e has been a renewed craze of herbal cosmetics and personal care products, especially in the skin care segment with the growing belief that chemical - based cosmetics are harmful and herbal cosmetics are safe being natural 1,2 . But since, it has been reported that the absorption of toxic metals through skin is very insignificant and can cause deleterious effects on our body 3,4 , there is an increase in scientific concern over the issue but unfortunately no sufficient data is available regarding the status of he rbal cosmetics for the presence of heavy metals except a few preliminary studies 5,6 . Thus the safety of these herbal cosmetics becomes doubtful and need further attention of the scientific community and the regulatory agencies. According to the World Hea lth Organization (WHO), heavy metals concentration of herbal medicines must definitely be controlled 4 . But WHO is silent regarding the maximum permissible limits of heavy metals in herbal cosmetics. In this case, Health Canada has taken the initiative and implemented a few measures to control heavy metal concentration in cosmetics and determined the maximum acceptable limits i.e Lead (10ppm), Arsenic (3ppm), Mercury (3ppm), Cadmium (3ppm) and Antimony (5ppm) 7 . Considering the importance of trace elements a nd toxic effects of heavy metals in herbal formulations and the fact that most of the scientific studies are confined to the herbal products meant for oral consumption 2,8,9 , it was imperative to screen the status of trace and heavy metals contents in herba l cosmetic products sold in Indian market. In the present study, 9 elements, Ca, Cu, Mg, Al, Zn, As, Hg, Pb and Cd were estimated quantitatively in 21 herbal cosmetic formulations by flame and hydride generation atomic absorption spectrometry followed by r ecovery studies as a part of method validation as per ICH guidelines. Material and Methods Samples: Twenty - one samples of marketed herbal cosmetic preparations were collected from local market of Delhi and Rohtak . The brand names were blinded and given the codes 1 to 21. The other details are presented in t able - 1. Instrument: Atomic absorption spectrophotometer (EC Electronics Corporation of India Limited AAS Element AS AAS4141) equipped with a deuterium lamp for background correction was used for determination of trace elements and Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 46 - 51 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 47 heavy metals. The hollow - cathode lamps for Al, Cu, Mg, Zn, Cd, Hg (ECIL) and Ca, As, Pb (Photron) were employed as radiation source. Hydride generator was used for hydride formation in As and Hg analysis. The flames used were air/acetylene and N 2 O /acetylene. Nitrogen was used as carrier gas. Chemicals: Nitric acid, hydrochloric acid, sulphuric acid, hydrogen peroxide, sodium borohydride and stannous chloride were of analytical grade (E . Merck). The water used in all experiment was ultrapure water obtained from Milli - Q - water purification system (Ranken Rion Ltd, India). The standard solutions were prepared in five different concentrations to obtain calibration curve by diluting stock sol utions (CPA Ltd) of 1000 ppm of each element immediately before use. Sample preparation: Samples were digested by the wet digestion method. 10 ml of nitric acid was added to 2 g of accurately weighed dried sample in a 100 ml beaker and was heated on a hot plate at 95°C for 15 min. The digest was cooled and 5 ml of concentrated nitric acid was added and heated for additional 30 min at 95°C. The last step was repeated and the solution was reduced to about 5 ml without boiling. The sample was cooled again and 2 ml of deionized water and 3 ml of 30% hydrogen peroxide was added. With the beaker covered, the sample was heated gently to start the peroxide reaction. If effervescence becomes excessively vigorous, sample was removed from the hot plate and 30% hydroge n peroxide was added in 1 ml increments, followed by gentle heating until the effervescence was subsides. 5 ml of concentrated hydrochloric acid and 10 ml of deionized water was added and the sample was heated for additional 15 min without boiling. The sam ple was cooled and filtered through a Whatman No. 42 filter paper and diluted to 50 ml with deionized water. Sample analysis: Digested samples were analyzed for Pb, Cd , Ca, Zn, Mg, Cu, and Al using flame atomic absorption spectrophotometer and for As, Hg using hydride generation technique. Hg was analyzed by cold vapour atomic absorption spectrometry. The 1000 ppm standard solutions of elements were diluted in five diff erent concentrations to obtain calibration curve for quantative analysis. All the measurements were run in triplicate for the samples and standard solutions. The instrumental conditions during the analysis of trace and heavy metals are listed in t able - 2 gi ving details about p arameters which are defined for respective metals. Recovery studies: The method of standard addition which is considered as a validation method 10 was used to demonstrate the validity of our method. Hence, a recovery test was performed using method of standard addition. Standard solutions containing Ca, Al, Mg, Cu, Zn, Pb, Cd, As and Hg were prepared and spiked with digested samples, after dilution of sample to 50 ml. Results and Discussion The contents of trace elements in the screen ed preparations as a mean of triplicate determination are described in t able - 3. The calcium concentrations varied from 152 to 16591 ppm, eight samples having contents between 2626 and 5985 ppm. Sample 3 had the lowest calcium concentration and sample 16 ha d the highest. Calcium was also found below detectable limit in total seven samples. The copper concentrations varied from 2.8 to 49.1 ppm, most samples having contents between 9.4 and 24.3 ppm. Sample 20 had the lowest copper concentration and Sample4 had the highest. The concentrations of copper were comparable in sample 6 and 7 with a range of 20.4 - 20.5, in 8 and 9 with a range of 21.9 - 22.2.The magnesium concentrations varied from 112 to 10,681 ppm, 12 samples having contents between 4737 and 8350 ppm wh ile three samples, 19, 20, and 21 had the equal magnesium concentration 112 ppm. Sample 9 had the highest magnesium concentration. The zinc concentrations varied from 4.8 to 56.57 ppm, most samples having contents between 31.4 and 56.57 ppm. Sample 20 had the lowest zinc concentration and sample 12 had the highest. The concentrations of zinc were comparable in sample 8 and 10 with a range of 50.46 -- 50.49, the same being true for sample code 18 and 11 at 46 and 46.12. The aluminium concentrations level range d from 126 to 5505 ppm, most samples having contents between 3900 and 5505 ppm. Sample 1 had the lowest aluminium concentration and sample 4 had the highest. Among the heavy metals, data presented in t able - 4 reveals that the mercury concentrations varied from 0.041 to 2.183 ppm, most samples having contents between 0.041 and 0.309 ppm. Sample 5 had the lowest mercury concentration and sample 18 had the highest. But mercury was found below detectable limit in eight samples. The concentrations of mercury we re comparable in samples 11 and 21 with a range of 0.072 - 0.076 ppm. According to the WHO, the permissible limit for mercury in herbal preparations is 1 ppm. In that way, two samples 9 (1.095 ppm) and 18 (2.183 ppm) were found to contain mercury concentrati on above permissible limit. The arsenic concentrations varied from 0.690 to 3.683 ppm, most samples having contents between 1.37 and 3.68 ppm. Sample 1had the lowest arsenic concentration and 16 had the highest. According to the WHO, the permissible limit for arsenic in herbal preparations is 10 ppm. All the herbal cosmetic products under investigation accumulated this metal at a level appreciably below the permissible limit.The cadmium concentrations varied from 0.625 to 1.875 ppm, most samples having cont ents between 0.625 and 1.09 ppm. Sample 19 had the lowest cadmium concentration and sample 14 had the highest. According to the WHO, the permissible limit for cadmium is 0.3 ppm in herbal preparations and unfortunately, all the herbal cosmetic products wer e found to contain cadmium concentration higher than the permissible limit.The lead concentrations level ranged from 1.470 to 33.1 ppm. Sample 4 had the lowest lead concentration and sample 21 had the highest. But lead was found below detectable limit in f our Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 46 - 51 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 48 samples. According to the WHO, the permissible limit for lead is 10 ppm. Six samples were contained Pb content above the permissible limit. The results of recovery study were within the acceptable range verifying the validity of proposed method for ana lysis (Table - 5) and revealed that any small change in the drug concentration in the solution could be accurately determined by the proposed method. In the present study, herbal cosmetic products were found to contain variable amounts of trace (nutrient) e lements. The variation in concentration of these elements may be mainly due to compositional differences of products and environmental condition where constituent plant is grown, use of fertilizer, pesticides. But generally it is concluded that the studied products are rich source of essential elements Mg, Ca, Zn, Cu, and Al and hence might play an important role in the maintenance of the skin nutritional requirements 11,12 . Toxic heavy metals Pb, Cd, As and Hg were detected in all the investigated cosmetic products. The cosmetic products related regulations do not decide maximum permissible limit values for heavy metals content in cosmetic products except of 1 ppm for Hg (ACSB 2007). However, Cd is prohi bited in any amount in cosmetics (Council Directive 76/768/EEC of 27 July 1976). The presence of heavy metals in cosmetics can cause serious problems to consumer as they can cause premature aging of the skin, skin allergies, and skin cancer. Further, toxic metals have a role to set up conditions that lead to inflammation in arteries and tissues, results in osteoporosis 13 . Thus, there is an urgent need for constant quality assessment of cosmetic products in the market in order to ensure the safety of consume rs. To achieve this, regulatory bodies and the government sector should implement the stringent policies to regulate and monitor the standards of herbal products manufactured, advertized, sold, and used. At the same time, scientific community should develo p simple and convenient analytical methods. The most widely techniques to analyze trace and heavy metals are atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP - MS), inductively coupled plasma atomic emission spectrome try (ICP - AES), and X - ray fluorescence spectroscopy (XFS) 14 . However, the instrumental methods of ICP - MS, ICP - AES, and XFS are usually more costly, and their use is not as straightforward and convenient as AAS. In this study, a simple, reliable, sensitive a nd convenient AAS method has been developed for quantitative estimation of trace metals and heavy metals which can conveniently be utilized for the quality control of herbal cosmetic preparations at industrial level. Conclusion 21 herbal cosmetic preparations sold in Indian market found to contain some biologically important trace elements, which may be helpful to impart therapeutic efficiencies u nfortunately, these products were also contained toxic heavy metal content above the permissible limits which may cause deleterious effects to the human health. In the present scenario, there is an urgent need to regulate them properly for the sustainable safety and efficacy. References 1. Kapoor V.P. Herbal cosmetic for skin and hair care , Nat. Prod. Radiance. 4(4) , 306 - 312 (2005) 2. Ernst E. Toxic heavy metals and undeclared drugs in Asian herbal medicines, Trends Pharmacol. Sc. 23 , 136 - 139 (2002) 3. Ayenimo J.G., Yusuf A.M., Adekunle A.S. and Makinde O.W. Heavy Metal Exposure from Personal Care Products, Bull. Environ. Contam. Toxicol. 84 , 8 – 14 (2010) 4. WHO. Environmental Health Criteria: International Programme on Chemical Safety. World Health Organization, Geneva. Pp 165 (1995) 5. Ajayi S.O., Oladipo M .O.A., Ogunsuyi H.O. and Adebayo A.O. , Determination of the minor and trace elements in Biriniwa tin pyrite and ornamental lead/zinc ore using neutron activation analysis, Bull. Chem. Soc. Ethiopia. 16(2) , 207 - 211 (2002) 6. Nanorom I.C., Igwe T.C. and Oji - Nnorom C.G. , Trace metal contents of facial (make - up) cosmetics commonly used in Nigeria, Afr. J. Biotechnol. 4(10) , 1133 - 1138 (2005) 7. Health Canada , Draft guidance on heavy metal impurities in cosmetics , Section 4 8. Nwoko C.O. and Mgbeahuruike L. , Heavy metal contamination of ready - to - use herbal remedies in south eastern Nigeria, Pak. J. Nutr. , 10(10) , 959 - 964 (2011) 9. Al - Omari S. , Determination of essential and toxic trace elements in ten herbal medicines using energy - dispersive XRF analysis , X - Ray Spectr om , 40 , 31 – 36 (2011) 10. Gomez M.R., Cerutti S., Somb L.L., Silva M.F. and Martınez L.D. , Determination of heavy metals for the quality control in Argentinian herbal medicines by ETAAS and ICP - OES , Food and Chem. Toxicol. , 45 , 1060 - 1064 (2007) 11. Ajasa A.M.O., Bellob M.O., Ibrahimb A.O., Ogunwandea I.A. and Olaworeb N.O. , Heavy trace metals and macronutrients status in herbal plants of Nigeria , Food Chemistry , 85(1) , 67 - 71 (2004) Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 46 - 51 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 49 12. Sharma D.K., Rai S., Arora S.S., Gupta P.M., Sharma R. and Chopra A.K. Study of t he trace elements in Aloe vera L. ( Aloe barbandensis Miller) viz. Liliaceae and its biological and environmental importance , J. Chem. Pharm. Res. , 3(3) , 64 (2011) 13. Farr G. , The hair tissue mineral analysis/why heavy metals are a hazard to your health , Updated 31Dec. , ( 2009 ) . Available from: ( http://www.becomehealthynow.com/ ebookprint.php?id=122 ) 14. Naithani V., Pathak N. and Chaudhary M. Evaluation of heavy metals in two major ingred ients of Ampucare , Int. J. Pharm. Sc. Drug Res. , 2(2) , 137 - 144 (2010) Table - 1 Details of herbal cosmetic products collected Sample code Constituents Use 1 Prunus armeniaca, Glycyrrhiza glabra, Avena sativa. Remove dark circles and blemishes. 2 Aloe barbadensis. Useful in skin darkness and dryness. 3 Ocimum sanctum. Antiseptic, antifungal, cleansing pores. 4 Carica Papaya. Heals damaged skin, control oil of skin. 5 Fragaria vesca. Remove skin dead cells and impurities. 6 Rosa, Trigonella foenum - graecum, Santalum album . Improve the complexion, skin pore cleansing and revitalizing. 7 Citrus sinensis . Astringent, moisturizer, scars, acne, smoothness. 8 Rosa, Santalum album . Antiaging, tone up the skin, tighten the pores. 9 Citrullus lanatus . Moisturizer, refreshner, sk in toner and tightener. 10 Solanum xanthocarpum. Antiaging, skin cleansing. 11 Citrus limon. Skin nourishment, cleansing and moisturizing. 12 Citrus sinensis , Carica papaya, Malus domestic , Prunus serotina, Fragaria vesca, Citrus limon, Punica granatum , Prunus persica . Pimples, antiaging, skin cleansing and softening. 13 Citrus limon, C rocus stivus, Curcuma longa, Santalum album. Depigmentation, skin cleansing and fairness. 14 Azadiracta indica. Antiaging, skin softening. 15 Rosa, Prunus amygdalus, C rocus stivus, Citrus limon, Curcuma longa, Santalum album. Fairness, soft skin. 16 Embelia ribes, Rubia cordifolia, Sida cordifolia, Acacia catechu, Valeriana jatamansi, Swarna gairik , Acorus calamus, Psoralea corylifolia, Aloe barbadensis, Curcuma longa, Azadiracta indica, Santalum album For acne, pimples and blemishes. 17 Eletteria cardamum, Valeriana jatamansi , Glycyrrhiza glabra, Vetiveria zizaniodes, Moringaoliefera, Cyperus scariosus, samudraphen, Brassica compestris, Valeriana wallichii, Saussurea lappa, Pavonia odorata , Cinnamomum glaucescens , Curcuma longa, Azadiracta indica, Santalum album Very efficacious in bringing new glows on the face. 18 Symplocos Racemosa , Punica granatum , Mangifera indica , Azadiracta indica. Fairness 19 Curcu ma longa, Santalum album. To prevents and cures skin infection, inflammation and blemishes. 20 Aloe barbadensis . Prevent ageing and dehydration of skin. 21 Citrus limon, Triticum stivum, Glycyrrhiza glabra, Calendula officinalis, Aloe barbadensis, Curcuma longa, Azadiracta indica, Santalum album To detoxify and nourish the skin. Protect skin from sun burn and pollutants. Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 46 - 51 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 50 Table - 2 Instrumental condition for analysis Element Current (mA) Slit width (nm) λ max (nm) Flame Color Flame Type AAS Technique Ca 3.5 0.5 422.7 Orange Air/C 2 H 2 Flame Mg 3.5 0.5 285.2 Orange Air/C 2 H 2 Flame Cu 5 0.5 324.7 Blue Air/C 2 H 2 Flame Zn 5 1.0 213.9 Blue Air/ C 2 H 2 Flame Al 10 0.5 309.3 Red Air/N 2 O/C 2 H 2 Flame Cd 3.5 0.5 228.8 Blue Air/C 2 H 2 Flame Pb 10 1.0 217 Blue Air/C 2 H 2 Flame As EDL 1 193.7 Blue Air/C 2 H 2 Hydride generation Hg EDL 0.5 253.6 - - Cold vapor AAS= Atomic absorption spectrometry Table - 3 Trace metal content in herbal cosmetic products (ppm) Sample Code Ca Cu Mg Zn Al Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD 1 2626±52.37 9.4±0.3 2963±90 51.23±0.40 126±7.94 2 5985±273.33 10.4±0.3 5108±97 50.57±0.28 4823±37.72 3 152±10.52 10.0±0.3 6300±43 49.76±0.39 5178±48.40 4 BDL 49.1±0.8 7351±87 51.67±0.57 5505±31.64 5 782±243.53 48.5±0.4 6983±714 50.06±0.12 5168±37.86 6 BDL 20.4±0.1 4737±33 35.66±6.99 3901±81.17 7 189±115.77 20.5±0.7 5146±159 39.65±0.30 4460±40.53 8 3206±315.05 21.9±0.3 4929±97 50.46±2.51 4511±89.61 9 3661±315.05 22.2±0.5 10681±900 54.01±0.14 4917±109.8 10 BDL 24.3±0.5 8112±481 50.49±0.58 4268±36.83 11 BDL 19.5±0.1 8350±307 46.12±0.26 4825±77.03 12 3633±199.57 22.0±0.6 7917±196 56.57±0.57 5311±66.58 13 4419±189.67 19.9±0.2 5316±101 50.78±0.41 4779±82.56 14 1489±115.66 22.9±0.2 8317±83 49.26±0.26 582±12.86 15 12222±42.72 23.2±0.5 475±38 18.69±0.40 313±32.01 16 16591±473.6 20.0±0.2 2071±566 21.11±0.24 1122±87.40 17 4217±307.73 19.2±0.2 1358±14 31.4±0.34 806±90.27 18 4292±265.86 16.2±0.6 3508±115 46±0.14 3780±95.09 19 BDL 14.1±1.9 112±0 6.89±0.09 325±69.50 20 BDL 2.8±0.3 112±0 4.8±0.15 275±55.75 21 BDL 4.3±0.3 112±0 6.6±0.00 230±21.63 Values are expressed as arithmetic mean ±SD ( n = 3). BDL = Below detectable limit. Research Journal of Chemical Sciences ______ _ _ _______________________________ ______________ _ ____ ISSN 2231 - 606X Vol. 2 ( 3 ), 46 - 51 , March (201 2 ) Res.J.Chem.Sci International Science Congress Association 51 Table - 4 Heavy metal content in herbal cosmetic products (ppm) Sample Code As Hg Cd Pb Mean±SD Mean±SD Mean±SD Mean±SD 1 0.690±0.026 BDL 0.725±0.100 8.450±0.050 2 1.370±0.036 BDL 0.792±0.208 6.750±2.091 3 1.467±0.025 0.118±0.004 1.235±0.115 12.850±0.577 4 2.147±0.035 0.168±0.007 0.792±0.289 1.470±0.852 5 1.843±0.035 0.041±0.007 0.892±0.252 7.400±1.975 6 1.637±0.029 BDL 0.992±0.321 10.040±1.158 7 2.013±0.006 BDL 0.785±0.115 6.750±0.563 8 2.263±0.021 0.311±0.012 0.825±0.100 11.300±1.988 9 2.927±0.047 1.095±0.031 0.858±0.343 8.400±1.302 10 3.077±0.191 0.065±0.010 0.792±0.231 6.430±1.975 11 2.637±0.038 0.072±0.014 0.725±0.173 9.350±2.963 12 3.213±0.040 BDL 0.758±0.153 9.390±3.438 13 2.927±0.047 BDL 1.258±0.551 12.030±3.995 14 1.557±0.042 BDL 1.875±0.499 6.750±2.479 15 2.237±0.038 BDL 1.092±0.252 BDL 16 3.683±0.181 0.309±0.021 1.158±0.208 BDL 17 2.923±0.045 0.157±0.021 0.692±0.115 BDL 18 3.423±0.045 2.183±0.026 0.992±0.321 BDL 19 1.887±0.021 0.278±0.011 0.625±0.000 16.630±3.992 20 1.430±0.035 0.083±0.010 0.658±0.058 20.580±3.746 21 1.563±0.038 0.076±0.010 0.625±0.000 33.100±1.975 Values are expressed as arithmetic mean ±SD ( n = 3). BDL = Below detectable limit. Table - 5 Recovery studies for trace elements and heavy metals Metal Base Value (ppm) Quantity Added (ppm) Quantity Found a (ppm) Recovery(%) b Al 126±7.94 10.0 135.78 97.8 Ca 3661±67.72 8.0 3668.72 96.5 Cu 4.33±0.22 3.0 7.24 97 Mg 112±0.30 1.0 112.96 96 Zn 18.70±0.4 2.0 20.63 96.5 As 2.926±0.047 0.3 3.224 99.33 Hg 1.094±0.030 0.2 1.292 99.00 Cd 0.858±0.34 1.0 1.85 99.20 Pb 8.400±1.30 5.0 13.356 99.12 Recovery test, a Mean value ( n = 3). b 100×[(found  base)/added].