7de vera-quirit-rodriguez.pmd Determination of Cr, Cd, Sn and Pb in Selected Herbal Products 82 SCIENCE DILIMAN (JULY-DECEMBER 2017) 29:2, 82-96 Determination of Cr, Cd, Sn, and Pb in Selected Herbal Products Available in Phil ippine Markets Joan S. De Vera University of the Philippines Diliman Leni B. Quirit University of the Philippines Diliman Irene B. Rodriguez* University of the Philippines Diliman Academia Sinica, Taiwan ABSTRACT The growing popularity of herbal products in the Philippines makes it i m p e r a t i v e t o m o n i t o r a n d e n s u r e s a f e t y o f c o n s u m e r s f r o m m e t a l contaminants. In this study, trace concentrations of Cr, Cd, Sn, and Pb in h e r b a l p r o d u c t s w e r e s i m u l t a n e o u s l y m e a s u r e d u s i n g a m i c r o w a v e - a s s i s t e d d i g e s t i o n a s s a m p l e p r e - t r e a t m e n t a n d i n d u c t i v e l y c o u p l e d plasma mass spectrometry (ICP-MS) for elemental detection. Using the o p t i m i z e d m e t h o d , r e c o v e r i e s o f E R M C D 2 8 1 , t h e p r i m a r y ce r t i f i e d reference material (CRM) used, were found to be between 80-89%, and the method detection limits (MDL) for Cr, Cd, Sn, and Pb were 0.15, 0.07, 0 . 3 , a n d 0 . 1 4 μg / L , r e s p e c t i v e l y. T h e l i n e a r r a n g e s f o r C r a n d o t h e r elements (Cd, Sn, and Pb) were 0.01-500 and 0.01-50 μg/L, respectively. All correlation coeff icients were 0.9999 or better. Most of the products t e s t e d h a d m e a s u r a b l e t r a c e m e t a l c o n c e n t r a t i o n s , w h i c h w e r e b e l o w the suggested maximum limits in herbal products. However, one product d e r i v e d f r o m m a n g o s t e e n e x c e e d e d t h e l i m i t f o r C d ( 0 . 4 2 m g / k g ) . Subsequent analysis of metal content in tea infusions showed that only a small fraction of metals may leach out, suggesting that consumption o f t e a i n f u s i o n s p o s e l e s s e r r i s k s . T h e o r d e r o f a b u n d a n c e o f m e t a l s f o u n d i n h e r b a l p r o d u c t s w a s C r > P b > C d > S n . T h e v a r i a b i l i t y o f m e t a l concentrations in herbal products underlines the fact that many plant ingredients are susceptible to contamination, and quality control during p r o c e s s i n g m u s t b e i m p r o v e d t o m i n i m i z e t h e p o s s i b i l i t y o f _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online Determination of Cr, Cd, Sn and Pb in Selected Herbal Products 84 accumulate in this organ (Hutton 1987; Satarug and Moore 2004; Navas-Acien et al. 2009). Pb also accumulates and affects the bones and liver (Hutton 1987). In addition, persistent low-level exposure to Pb can cause neurotoxic effects to children because of the permeability of their blood brain barrier to Pb (Jarup 2003). No guideline values have been set for Cr and Sn despite the known serious health conditions associated with these two metals. Some forms of Cr and Sn, such as Cr (VI) and organotin compounds, are considered toxic for humans (Kimbrough 1976; Kotaœ and Stasicka 2000; Yu et al. 2008). In consideration of the known risks of these metals, they should be monitored in herbal products, especially because these products are sold as supplements and are regularly consumed for a long period of time. This work is aimed towards the development and optimization of a method suitable for the determination of metal concentrations in herbal products commonly sold and consumed in the Philippines. An analytical method was optimized and validated using the certif ied reference materials (CRM) and spiked samples prior to the application of the method to actual samples. The data from this study may serve as a background on the extent of metal contamination in herbal products in the country since publications regarding metal levels are scant. MATERIALS AND METHODS The following CRM were used for the optimization and validation of the sample preparation and analytical method: ERM-CD281 (rye grass obtained from Institute for Reference Materials and Measurements, Belgium), BCR 150 (skim milk powder from Institute for Reference Materials and Measurements, Belgium), DORM-3 (f ish protein from National Research Council, Canada), and NIST 1643e (Trace elements in water, National Institute of Standards and Technology, USA). ERM-CD281 was the primary CRM used because it has the closest matrix as the samples. BCR 150 and DORM-3 were analyzed to check the applicability of the method to more complex sample matrices. NIST 1643e was used as an additional CRM for tea infusions because it was the available reference material that has the closest matrix to liquid samples. For further validation, the applicability of the sample pre-treatment scheme for liquid samples was evaluated following the standard addition method on one of the herbal liquid samples (Noni plant juice), which was spiked with known analyte concentrations between 0.67 μg/L and 13.33 μg/L. A total of 33 commonly available herbal products were purchased from local drug stores and supermarkets in Quezon City, Philippines between September 2011 and J.S. De Vera, et al. 85 November 2011. All products analyzed were manufactured in the Philippines and made from plant ingredients that are endemic to the country except for the Noni juice sample, which was included in the study to serve as the matrix for standard addition validation. The products were chosen according to their availability and whether consumers can easily acquire them through over-the-counter purchases and without prescription. The plant ingredients, formulations, and purported health benef its of the herbal products included in the study are summarized in Table 1. Of the samples analyzed, four plants, namely lagundi, sambong, amplaya and garlic, are listed in the circular of the Department of Health (1995) as safe, effective and scientif ically-validated herbal medicinal plants. Malunggay, ginger, mangosteen, coconut and papaya plants are listed as medicinal plants with folkloric basis, and thereby require fur ther scientif ic validation (Department of Health 1995). Other plants that were not mentioned in the list are also believed to have medicinal values based on folkloric evidences. Herbal products made from lagundi were already approved as drugs to cure cough, while other lagundi-derived products are marketed as dietary supplements. Plant Ingred ient (Scientific Name) Formulations Common Med icinal Benefits Ampalaya (Momordica charantia) capsule, tea anti-diabetes Banaba (Lagerstroemia speciosa) capsule, tea good remedy for diabetes, high blood pressure and kidney problems Coconut (Cocos nucifera) Oil f ights viral, bacterial, fungal and protozoan infections; aids in digestion and nutrient absorption Garlic (Allium sativa) capsule anti-cholesterol Ginger (Zingiber officinale) tea relief for gaseous distention, cough, arthritis and wounds Lagundi (Vitex negundo) capsule, syrup, tea anti-cough, asthma relief Malunggay (Moringa oleifera) capsule, coffee relief for arthritis, scabies, wounds and constipation Mangosteen (Garcinia mangostana) capsule, tea relief for diarrhea and stomach pain; contains anti-oxidant Narra (Pterocarpus indicus) capsule immune system support Noni (Morinda citrifolia) syrup anti-oxidant; supports immune system; has tumor-f ighting properties Sambong (Blumea balsamifera) capsule, tea diuretic Spirulina (Arthrospira) capsule antioxidant, anti-inflammatory and detoxif ier. Turmeric (Curcuma longa) capsule relief for digestive disorders Papaya (Carica papaya) syrup relief for constipation and wounds Table 1. Plant ingred ients, formulations and common med icinal benefits of the herbal products included in this study Determination of Cr, Cd, Sn and Pb in Selected Herbal Products 86 For the solid samples, tea capsules/tea bags/coffee sachets were pooled and mixed to achieve sample homogeneity. From the pooled sample, triplicate samples (0.2500 g weighed to the nearest 0.0001 g) were obtained and digested using a Multiwave 3000 microwave system (Anton Paar, Austria). For samples in liquid form, triplicate aliquots (1 mL) were obtained for digestion. The digestion mixture is composed of 2 mL concentrated superspure HNO 3 , 1 mL concentrated superspure HCl, and 5 mL ultrapure water. The digestion program for the complete mineralization of samples was as follows: digestion was initially carried out at 250 W for 1 minute; then ramped to 400 W for 5 minutes; 600 W for 5 minutes; and, 800 W for 20 minutes. The digestion system was allowed to cool before the digested samples were transferred to polyethylene containers, bulked to 15 mL with ultrapure water, and subsequently f iltered through 0.45 μm Nylon f ilters prior to elemental analysis. The concentrations of metals in tea infusions were also measured. For every brand of tea, three infusions prepared from different tea bags obtained from the same batch were analyzed. Tea infusion was made by adding 40 mL boiled (~100 oC) ultrapure water to a tea bag. The infusion was kept for 3 minutes, which is the commonly suggested length of infusion when preparing tea drinks. The infusion was f iltered through a 0.45 μm Nylon f ilter, mixed with 0.5 mL concentrated HNO 3 , and bulked to 50 mL with ultrapure water. Because of the formation of precipitates upon acidif ication, the infusions were f iltered again with new Nylon f ilters prior to elemental analysis. Simultaneous analyses of Cr, Cd, Sn and Pb in acid-digested sample solutions and tea infusions were performed using an Agilent 7500cx ICP-MS (Agilent, Germany) equipped with a MicroMist glass concentric nebulizer and an integrated autosampler (I-AS with type A vials, 89 x 6 mL capacity). Calibration standards ranging between 0.01 μg/L and 500 μg/L were prepared from stock standard solutions of Cr, Cd, Sn and Pb (10000 μg/mL, CPI Technology, USA). For Cr, the linear range was established from 0.01 μg/L to 500 μg/L. A wider range of concentration was necessary since ERM CD281 and the samples contain relatively higher amount of Cr. The linearity for Cd, Sn and Pb were set at a much narrower range of concentration (0.01 μg/L - 50 μg/L). The correlation coeff icients (r2) for all the calibration curves were 0.9999 or better. All solutions analyzed were spiked on-line with internal standard mixture containing Ge (1 mg/L), In (10 mg/L), and Re (10 mg/L) for signal drift correction. In addition, intra- and inter-day variabilities of the method were assessed by repeated measurements of a solution containing a f inal concentration equivalent to 1 ppb of each analyte and ERM CD281-digested solutions, respectively. The detection limit was estimated by analyzing seven individually prepared solutions containing 1 μg/L of each analyte in 1% HNO 3 , which were subjected to digestion and subsequent J.S. De Vera, et al. 87 ICPMS analysis. The detection limit was only estimated for aqueous digests and not for solid samples because of the diff iculty of ensuring homogeneity of spiked standards in solid samples. The standard deviations of the seven measurements multiplied with the student’s t-value (i.e. , student’s t-value for n = 7 at 99% conf idence level is 3.143) represents the estimated detection limit described in EPA document 40 CFR part 136. RESULTS AND DISCUSSION A prerequisite to trace metal analysis in actual samples is the existence of an analytical method that is optimized for the specif ic measurement and duly validated to be suitable for the deemed purpose. Prior to the analysis of herbal products, the reliability of the method was established by analyzing CRM and spiked liquid herbal product. The results from the analysis of the different CRM are summarized in Table 2. Results from the analysis of the primary CRM, ERM-CD281 (n = 15), revealed that the recoveries for Cr, Cd, Sn and Pb were at least 80%. Results from other solid r e f e r e n c e m a t e r i a l s D O R M - 3 a n d B C R 1 5 0 s h o w e d t h a t m e a s u r e d v a l u e s approximate well the cer tif ied values of the analytes, indicating that the mineralization procedure and subsequent elemental determination were robust for the determination of more complex sample matrices. This is particularly advantageous for the samples in this study which were of complex nature. Results of the analysis of NIST 1643e revealed that the optimized method was suitable for the analysis of Cr, Cd and Pb as reflected by the agreement between certif ied and measured values. To further validate the suitability of the method for liquid samples, as tea infusions may have more complex matrix than water, we also carried out the standard addition Table 2. Val idation of d igestion method and ICPMS detection in CRM (mean ± SD, n = 15 for ERM-CD281, n =3 for other CRM) ERM- Certif ied Value 24.8 ± 1.3 0.120 ± 0.072 0.062 ± 0.011 1.67 ± 0.11 CD281 Measured Value 19.8 ± 0.5 0.101 ± 0.003 0.067 ± 0.003 1.46 ± 0.03 DORM-3 Certif ied Value 1.89 ± 0.17 0.290 ± 0.020 0.066 ± 0.012 0.39 ± 0.05 Measured Value 2.08 ± 0.10 0.27 ± 0.01 0.08 ± 0.03 0.33 ± 0.01 BCR 150 Certif ied Value not certif ied 0.0218 ± 0.014 not certif ied 1.00 ± 0.04 Measured Value 0.018 ± 0.001 0.76 ± 0.01 NIST Certif ied Value 20.4 ± 0.24 6.568 ± 0.073 not certif ied 19.63 ± 0.21 Measured Value 21.4 ± 0.4 6.7 ± 0.1 20.2 ± 0.7 Certified Reference Material Cr mg/kg Cd mg/kg Sn mg/kg Pb mg/kg Certified Reference Material Crμg/L Cdμg/L Snμg/L Pbμg/L Determination of Cr, Cd, Sn and Pb in Selected Herbal Products 88 method (Table 3). One of the liquid samples, Noni juice, was spiked with mixtures containing different concentrations of the target analytes. Except for Cr at the lowest spiked concentration, most of the analytes at different concentrations resulted in acceptable recoveries within the range of 90% to 120%. The high standard deviation observed in the values obtained for Cr at the lowest spiked concentration may have been due to the diff iculty in completely mineralizing Cr after it has equilibrated with the Noni juice. Evaluation of the method detection limits (MDL) following the suggested protocol by the US Environmental Protection Agency resulted in the following values: 0.15, 0.07, 0.3, and 0.14 μg/L for Cr, Cd, Sn and Pb, respectively. These values were low enough to allow trace determination of the analytes should they be present as contaminants in the target samples. Digested samples and tea infusions with metal concentrations below MDL were reported as Pb > Cd > Sn. For Cr, four samples obtained concentrations that were below the MDL. In solid samples, including tea leaves, coffee powder, and capsules, Cr was determined to be within the range of 0.14 to 2.6 mg/kg. In liquid samples, including juice and oils, the values ranged from 50 to 1100 μg/L. We observed that the values obtained for Cr are relatively higher than what Solidum et al. (2012) measured in Philippine herbal products, wherein only 3 out of 10 products have measurable Cr concentrations that range between 0.0084 and 0.0578 mg/kg. The Cr concentrations measured in this study are comparable to values (0.34–2.12 mg/kg in herbs) reported in a study performed in Turkey (Baº gel and Erdemo lu 2006). However, compared to the results obtained in studies done in Pakistan (11.4–66.6 mg/kg) and Poland (0.3–63.1 mg/kg), the samples in the present study have much lower concentrations of Cr (Leœniewicz et al. 2006; Mahmood et al. 2013). The detection of Cr in most samples warrants further studies that focus on speciation studies that attempt to f ind out whether Cr is present in these products as Cr (VI), which poses a higher health risk compared to Cr (III). Pb, another ubiquitous contaminant in the environment, was determined in 30 samples and was below the detection limit in three samples. The concentrations were within the range 0.02–1.2 mg/kg and 5–51 μg/L in solid and liquid products, respectively. Although none was found to exceed the 10 mg/kg guideline value, the determined values suggest that some products are susceptible to Pb contamination at signif icant concentrations. Pb is known to be bio-accumulated, and since most of these products are taken regularly, presence of Pb at trace 24.8 24.9 19.4 22.8 19.6 19.8 (1.3) (0.9) (0.6) (0.3) (0.2) (0.5) 0.12 0.098 0.086 0.099 0.096 0.101 (0.007) (0.008) (0.003) (0.001) (0.002) (0.003) 0.062 0.071 0.055 0.04 0.049 0.067 (0.011) (0.004) (0.009) (0.01) (0.008) (0.003) 1.67 1.38 1.18 1.42 1.24 1.46 (0.11) (0.01) (0.05) (0.02) (0.04) (0.03) Cr Cd Sn Pb Measured concentration (mg/kg) and standard deviation of measurements (SD) Day 3 Day 4 Day 5Day 1 Day 2 Certified Values Element Table 4. Inter-batch repeatabil ity study using ERM CD281 measured during five d ifferent analyses days (n = 3 per day of analysis) ğ  Determination of Cr, Cd, Sn and Pb in Selected Herbal Products 90 concentrations may lead to health risks. In the measurements made by Solidum (2014) for Pb, medicinal plants and herbal tea ingredients in the Philippines were in the range of 0.006–0.873 mg/kg, which is comparable to this study. Compared to herbal products from other countries, the levels of Pb measured in the Philippines (e.g. , this study and Solidum 2014) were lower. For instance, some samples exceeded the 10 mg/kg limit in studies performed in Pakistan (0.5–41.01 mg/kg) by Mahmood et al. (2013) and in Malaysia (2.34–13.2 mg/kg) by Ang (2008). The coffee in this study was also found to have lower Pb concentration (0.02 mg/kg) than that of the study made by Kapur and West (1974) in England (0.45–1.65 mg/ kg). A1 Ampalaya Capsule 2.6 ± 0.2 0.127 ± 0.002 < MDL 0.33 ± 0.02 A2 Ampalaya Capsule 0.98 ± 0.04 0.059 ± 0.001 < MDL 0.6 ± 0.1 A3 Ampalaya Tea leaves 0.8 ± 0.2 0.052 ± 0.001 0.059 ± 0.003 0.58 ± 0.03 A4 Ampalaya Tea leaves 0.25 ± 0.01 0.013 ± 0.001 0.010 ± 0.003 0.13 ± 0.02 B1 Banaba Capsule 0.9 ± 0.1 0.187 ± 0.004 < MDL 1.0 ± 0.3 B2 Banaba Tea leaves 0.8 ± 0.1 0.223 ± 0.008 0.054 ± 0.004 0.19 ± 0.01 B3 Banaba Tea leaves 0.7 ± 0.1 0.187 ± 0.003 0.027 ± 0.003 0.19 ± 0.02 G1 Garlic Capsule 0.43 ± 0.05 0.074 ± 0.004 < MDL 0.2 ± 0.1 R1 Ginger Tea leaves 4.2 ± 0.2 0.057 ± 0.001 0.013 ± 0.004 0.11 ± 0.02 L1 Lagundi Capsule 1.1 ± 0.3 0.011 ± 0.001 < MDL 0.5 ± 0.1 L2 Lagundi Capsule 0.6 ± 0.1 0.11 ± 0.01 < MDL 0.20 ± 0.03 L3 Lagundi Tea leaves 0.58 ± 0.04 0.010 ± 0.002 0.035 ± 0.001 0.19 ± 0.01 L4 Lagundi Tea leaves 0.6 ± 0.1 0.014 ± 0.002 0.028 ± 0.001 0.15 ± 0.01 M1 Malunggay Capsule 0.5 ± 0.1 0.03 ± 0.01 0.04 ± 0.01 0.30 ± 0.03 M2 Malunggay Capsule < MDL 0.025 ± 0.001 0.07 ± 0.01 0.71 ± 0.01 M3 Malunggay Coffee 0.16 ± 0.04 < MDL 0.003 ± 0.001 0.02 ± 0.01 M4 Malunggay Tea leaves 0.8 ± 0.1 0.007 ± 0.001 0.02 ± 0.01 0.12 ± 0.04 M5 Malunggay Tea leaves 0.14 ± 0.02 0.012 ± 0.001 0.029 ± 0.003 0.15 ± 0.02 T1 Mangosteen Capsule 0.21 ± 0.03 0.083 ± 0.001 earned her BS and MS Chemistry degrees from the IC-UPD. This work comprised her MS thesis. JS De Vera is currently a Ph.D. candidate at the Department of Earth Sciences of the University of Toronto, Canada. Leni L. Quirit is a professor at the IC-UPD with specializations in analytical chemistry, radiochemistry, and nuclear chemistry. LL Quirit earned her Ph.D. Chemistry from the Ateneo De Manila University. Irene B. Rodriguez is currently a postdoctoral fellow at the Academia Sinica in Taiwan. IB Rodriguez earned her doctoral degree in Chemistry from the Karl-Franzens University of Graz in Austria.