- Email: [email protected]
Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
International Journal of Mass Spectrometry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Young Scientist Feature
Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species Fransiska Dewi, Lay Peng Sim, Juan Wang, Benny M.K. Tong, Richard Y.C. Shin ∗ , Tong Kooi Lee Chemical Metrology Laboratory, Chemical Metrology Division, Applied Sciences Group, Health Sciences Authority of Singapore, 1 Science Park Road, #01-05/06, The Capricorn, Singapore Science Park II, Singapore 117528, Singapore
a r t i c l e
i n f o
Article history: Received 20 September 2016 Received in revised form 1 November 2016 Accepted 2 November 2016 Available online xxx Keywords: Certified reference material Apple juice Toxic elements Arsenic speciation Isotope dilution mass spectrometry Standard addition
a b s t r a c t In the development of the apple juice certified reference material, isotope dilution mass spectrometry method was used for the assignment of certified values and long-term stability study for cadmium and lead. For total arsenic, inorganic arsenic, as well as dimethylarsenic acid, standard addition method was used. The analytes were found to be homogenous and stable over a period of at least 4.5 months at storage temperature of −20 ◦ C (long-term stability). The certified mass fraction values were (0.220 ± 0.011) mg/kg for cadmium, (0.245 ± 0.014) mg/kg for lead, (0.185 ± 0.015) mg/kg for total arsenic, (0.124 ± 0.012) mg/kg for inorganic arsenic [As(III)+As(V)] and (0.0601 ± 0.0052) mg/kg for DMAA. The recovery of the arsenic species was over 99%. This apple juice certified reference material can be used for method validation or as a quality control material by routine testing laboratories. © 2016 Elsevier B.V. All rights reserved.
1. Introduction For drinking water, the maximum contaminant level for cadmium has been set at 3 g/L according to the World Health Organization (WHO) and 5 g/L according to the US Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA) [1–3]. FDA has also established a guidance level of 50 ppb of lead in ready-to-drink fruit juices, in line with the Codex Alimentarius Commission standard [4,5]. In addition, FDA has proposed an “action level” for inorganic arsenic in apple juice to be 10 g/L [6]. It is the same level as for arsenic in drinking water set by the EPA [3]. Meanwhile, the Agri-Food and Veterinary Authority of Singapore (AVA) has regulated the maximum amount of lead and arsenic in fruit juices to be 300 and 200 ppb, respectively [7]. There is no limit specified for cadmium in fruit juices. However, the Sale of Food Act mentions that natural mineral water and any article of food shall not contain more than 10 ppb and 200 ppb of cadmium, respectively [7]. Cadmium is considered a cumulative nephrotoxicant, whose level in human organs increases with age due to the lack of an active biochemical process for its elimination coupled with renal
∗ Corresponding author. E-mail address: Richard [email protected] (R.Y.C. Shin).
reabsorption. There has been accumulating evidence for the carcinogenic risk of chronic cadmium exposure [8]. Lead, on the other hand, has the ability to affect almost every system in the human body, including the reproductive, neurological, hematopoietic, hepatic and renal systems [9]. Long term low level exposure in children is harmful for the brain and nervous system [10]. As in cadmium and lead, acute and chronic exposures to arsenic can also cause adverse health effects to human including dermal changes, damage to internal organs and carcinogenic effects [11,12]. Arsenic found in fruits and vegetables is primarily organic arsenic and only less than 10% of the arsenic is present in the inorganic form [11]. Inorganic arsenic in apple juice may come from processing aids, prior and/or current use of pesticides containing arsenic on apple orchards, natural arsenic in soil or water and atmospheric deposition from industrial activities [13]. In human, organoarsenic compounds including monomethylarsinic acid (MMAA) and dimethylarsinic acid (DMAA) are not readily taken up by the cell, subjected to limited metabolism and excreted close to their original form in the urine [11]. In contrast, the soluble inorganic arsenic can be absorbed and accumulated in tissues and body fluids. Hence, inorganic arsenic species are more toxic than the organic ones and As(III) is more toxic than As(V) [11,12]. Arsenic speciation gives valuable information that helps in judging the actual level of toxicity.
http://dx.doi.org/10.1016/j.ijms.2016.11.001 1387-3806/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
2
This paper describes the development of an apple juice certified reference material (CRM) containing cadmium, lead, inorganic [As(III) and As(V)] as well as organoarsenic (DMAA) species. The development of the CRM, including procedures for sample preparation, homogeneity study, stability study, assignment of reference values and uncertainty evaluation, was carried out in accordance with the requirements of ISO Guides 34 and 35 [14,15]. Prior to establishing the certified mass fraction values for the analytes in the material, a method development was conducted using CRMs from the National Metrology Institute of Japan (NMIJ) (Tsukuba, Japan). The results obtained were in good agreement with certified concentration levels. To the best of our knowledge, there are currently no available CRMs for arsenic speciation in fruit juice matrix. Hence, rice flour CRMs were used in the method development. 2. Material and methods 2.1. Reagents, standard solutions and CRMs All experimental work was carried out in class 100 low laminar flow fumehoods or a class 10,000 clean room. Standard and sample solutions were prepared gravimetrically on a microanalytical balance (Mettler Toledo XP205 or XP26, Greifensee, Switzerland). The certified reference standards for cadmium, lead and total arsenic were obtained from the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). Isotopically enriched isotopes were purchased from Oak Ridge National Laboratory (Oak Ridge, TN, USA). For determination of cadmium, lead and total arsenic, all working solutions were diluted with 5% HNO3 (aq). Nitric acid (67–70% HNO3 , Ultrapur-100, Kanto Kagaku Singapore Pte Ltd) used was distilled twice using DST-1000 sub-boiling distillation system (Savillex Corporation, MN, USA). The As(III), As(V) and DMAA standards were obtained from the National Metrology Institute of Japan (NMIJ) (Tsukuba, Japan). Stock solution for As(III) was prepared by dissolving As(III) trioxide powder in 1% NaOH (aq), while stock solution for As(V) and DMAA were prepared by diluting with Milli-Q element water (18.2 M cm, Millipore Corporation, MA, USA). The certified reference standards and isotopically enriched isotopes used are listed in Table 1. The brown rice (CRM 7532-a) and white rice (CRM 7503-a) CRMs used for method development were obtained from NMIJ. The high-performance liquid chromatography (HPLC) buffer solution used for arsenic speciation was prepared by adding 0.7 g tetramethylammonium hydroxide pentahydrate (99%, Aik Moh Paints and Chemicals, Singapore), 1.6 g sodium 1-butanesulfonate (≥ 99.0%, Sigma Aldrich, MO, USA), 0.7 g malonic acid (Reagentplus 99%, Sigma Aldrich, MO, USA) and 0.5 mL methanol (HPLC grade, J.T. Baker, PA, USA) into 1 L of Milli-Q water. The pH of the buffer solution was approximately 3.0. 2.2. Instrumentations An Agilent Technologies 7700x ICP–MS system (ICP–MS) (Agilent Technologies International Japan, Ltd., Tokyo, Japan) equipped with an octopole collision cell was used for measurements of cadmium and lead. Measurements of total arsenic were carried out using a Finnigan Element 2 (Finnigan MAT GmbH, Bremen, Germany) sector-field ICP–MS (SF–ICP–MS) using a self-aspirating PFA MicroFlow nebulizer. For arsenic speciation, an Agilent Technologies 1200 HPLC was coupled with the ICP–MS system to separate and measure the different arsenic species [As(III), As(V) and DMAA]. The HPLC was equipped with an automatic sample injector for direct sample introduction into the ICP–MS. The instruments were conditioned and optimized daily to achieve optimum
sensitivity and stability. The typical operating conditions and data acquisition parameters are summarized in Table 2. 2.3. Preparation of candidate CRM sample The apple juice material, obtained from a local supermarket, was screened and found to contain negligible amount of cadmium, lead and arsenic. The material was filtered using Nalgene RapidFlowTM Filter Units (90 mm nylon membrane, 0.2 m pore, VWR, PA, USA), preserved with sodium benzoate (1.2 g/L, >98.0%, HPLC grade, Tokyo Chemical Industry, Tokyo, Japan) and spiked with cadmium nitrate tetrahydrate, lead (II) nitrate from Sigma Aldrich (MO, USA) as well as As(III) trioxide, As(V) and DMAA from NMIJ. The mixture was then homogenized by mixing on a shaker for 1 h and bottled into 125-mL high-density polyethylene (HDPE) bottles. Each bottle containing 100 mL of apple juice was flushed with nitrogen gas for 15 s before sealing and labelled according to its dispensing sequence number. A total of fifty four bottles were prepared. The targeted ranges of concentration for cadmium, lead, inorganic arsenic and DMAA in the apple juice were 0.2–0.4 mg/kg, 0.2–0.4 mg/kg, 0.02–0.2 mg/kg and 0.01–0.1 mg/kg, respectively. The apple juice was not acidified so that it would closely match in terms of matrix, measurands and concentrations to the type of materials encountered in routine testing or calibration. For long term storage, the material was stored in frozen state at −20 ◦ C to reduce the risk of deterioration. 2.4. Homogeneity study, stability study and assignment of reference values The homogeneity of the material was established by selecting six bottles using a stratified random sampling scheme. The samples were analyzed by ICP–MS using isotope dilution mass spectrometry (IDMS) for cadmium and lead, by SF–ICP–MS using standard addition method for total arsenic and by HPLC–ICP–MS using external calibration method for arsenic species. The homogeneity of each analytes was tested using the one-way analysis of variance (ANOVA) on duplicate results from these six bottles. The result from the homogeneity study of total arsenic was also used for assignment of reference value. For the assignment of reference values three bottles were randomly selected and two subsamples were taken from each bottle. The measurements were carried out using IDMS (cadmium and lead) and standard addition (arsenic species) methods. The results were taken as the first point (t = 0 month) in the long-term stability study. Two more stability points were determined at time interval of 3 and 4.5 months, using the classical design [16]. 2.5. Sample preparation for analysis For determination of cadmium, lead and total arsenic content, a multiple spiking approach was used for the sample preparation. Calculated amounts of enriched isotope spike solutions (111 Cd and 206 Pb) were added gravimetrically into approximately 1 g of apple juice. The mixture was digested with 2.5 mL of concentrated HNO3 at room temperature overnight. Afterwards, a calculated amount of gallium (Ga) standard solution (SRM 3119a, NIST, Gaithersburg, MD, USA) was added into the digest and the mixture was diluted to 50 g using Type I Milli-Q water. The final weight of the diluted digest was recorded. The digest was then used to prepare sample blends (cadmium and lead) as well as unspiked and spiked solutions (total arsenic). This protocol was employed for the homogeneity testing, assignment of reference values as well as long-term stability studies. For arsenic speciation, approximately 0.5 g (for external calibration) or 1 g (for standard addition) of apple juice was diluted with
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
3
Table 1 Certified reference standards used. Analytes
Certified Reference Standard
Cadmium Lead Arsenic Gallium As(III) As(V) DMAA a b
Isotope Spike (Isotopic Fraction)
Code
Certified Value
SRM 3108 SRM 3128 SRM 981a SRM 3103a SRM 3119ab CRM 3003-a CRM 7912-a CRM 7913-a
(10.007 ± 0.027) mg/g (9.995 ± 0.014) mg/g – (9.999 ± 0.015) mg/g (10.00 ± 0.04) mg/g (100.001 ± 0.018) mg/kg (99.53 ± 1.67) mg/kg (25.11 ± 0.70) mg/kg
111 206
Cd (96.44%) Pb (99.76%)
– – – – – –
Used for mass bias correction. Used as an internal standard.
Table 2 Operating conditions for ICP–MS, SF–ICP–MS and HPLC instruments. ICP-MS (Agilent 7700x) Element
Cadmium
RF power/W Carrier gas flow rate/L min−1 Make up gas flow rate/L min−1 Sampling depth/mm Spray chamber temperature/◦ C Ion lens settling Helium gas flow/mL min−1 Data acquisition integration time/s Points per peak Repetitions m/z
4.7
97 (Mo); 111, 112, 114 (Cd); 118 (Sn)
Lead 1550 0.9 0.3 8 2 Optimized daily 4.7 3 3 3 202 (Hg); 204, 206, 207, 208 (Pb)
Arsenic (speciation)
3.0
35 (Cl); 75 (As)
SF-ICP-MS (Element 2) Element
Arsenic
RF power/W Scanning mode Settling time/ms Resolution Sample time/ms Samples per peak Mass window Runs Passes Total time per sample/min m/z
1250 E-scan 1 High (10,000) 100 20 100 5 6 2.01 69, 71 (Ga); 75 (As)
Agilent 1200 HPLC Element
Arsenic (speciation)
Column Mobile phase Temperature/◦ C Flow rate/mL min-1 Total elution time/min
L-ODS or C18 MG-S5 (4.6 ID x 150 mm, 5 m, Waters type, Chemical Evaluation and Research Institute, Japan) 10 mM sodium 1-butanesulfonate/4 mM malonic acid/4 mM tetramethylammonium hydroxide/0.05% methanol in type I water (pH 3.0) 30 0.5 6
Type I Milli-Q water to approximately 10 g. For homogeneity study using external calibration, no further dilution was required before sample analysis. For the assignment of reference values and longterm stability studies using standard addition, the diluted sample was used to prepare unspiked and spiked solutions. The samples were filtered through a 0.2 m filter (regenerated cellulose, 15 mm, Agilent, CA, USA) and transferred into crimp snap polypropylene vials (Agilent, CA, USA) for analysis using HPLC–ICP–MS.
2.6. Analytical methods—exact-matching IDMS, standard addition multi-point calibration and standard addition by intensity ratio
spikes were added into the sample and standard solutions. Total arsenic content in apple juice was determined using standard addition method by measuring intensity ratio of arsenic to gallium (internal standard) [19]. An unspiked solution and four spiked solutions were prepared from each diluted digest. The analysis of arsenic species in apple juice was performed using multi-point calibration standard addition technique [20]. Different amounts of arsenic species standard solution were spiked into the sample to produce an unspiked and four spiked solutions. The details of the methods can be found in our earlier publications [21–23]. At least two reagent blanks were also prepared by subjecting them to all sample preparation steps to evaluate possible blank contributions. All measured intensity ratios were subjected to blank correction before the concentration of each analyte was calculated.
Exact-matching IDMS method [17,18] was employed in the measurement of cadmium and lead. Known amounts of isotopic Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
4 Table 3 LODs and LOQs of As(III), As(V) and DMAA. Analytes
LOD (mg/kg)
LOQ (mg/kg)
As(III) As(v) DMAA
0.00041 0.00017 0.00029
0.00137 0.00056 0.00096
3. Results and discussion 3.1. Method development using brown rice (CRM 7532-a) and white rice flour (CRM 7503-a) Since there are no commercially available fruit juice CRM for arsenic speciation, rice flour CRMs (CRM 7532-a and CRM 7503a) were used for the method development. The different type of arsenic species [As(III), As(V) and DMAA] are also commonly found in rice flour. Unlike apple juice, arsenic speciation in rice flour requires an extraction procedure. Water extraction (for CRM 7503a) or acid extraction using diluted HNO3 (1% v/v) (for CRM 7532-a) was carried out using microwave-assisted digestion method (95 ◦ C for 90 min) [24–26]. The mass fractions of As(III), As(V) and DMAA were determined by using multi-point calibration standard addition method. The arsenic species were separated by HPLC and subsequently measured by ICP-MS. Table 3 shows the limit of detections (LODs) and the limit of quantifications (LOQs) of the method for the different species [27]. Since As(III) and As(V) can undergo inter-conversion during extraction, the mass fraction of the total inorganic arsenic [As(III) + As(V)] are usually reported, instead of the individual species. Good agreement between the measured and certified values was observed for both inorganic arsenic and DMAA (see Table 4). Hence, the developed method was deemed suitable and was adopted for the quantification of the arsenic species in apple juice.
3.2. Homogeneity and stability studies Homogeneity testing was performed on six bottles with two sub-samples taken from each bottle. No significant differences in the between and within-bottle variances were found using oneway ANOVA at 95% confidence level [14]. Thus, the material was regarded to be sufficiently homogeneous. The relative standard uncertainty due to between-bottle inhomogeneity (ubb ) were 0.20% for cadmium, 0.28% for lead, 0.52% for total arsenic, 1.16% for inorganic arsenic [As(III) + As(V)] and 0.99% for DMAA. The long-term stability of the analytes in apple juice was assessed over time interval of 3 and 4.5 months. Two subsamples from two bottles of the material were analyzed at each time point. The analytes in apple juice were shown to be stable over a period of at least 4.5 months (see Fig. 1). The relative standard uncertainties due to long-term storage (4.5 months or approximately 20 weeks) of the material (ults ) estimated from the standard error of the slope were 0.47% for cadmium, 0.55% for lead, 0.76% for total arsenic, 0.40% for inorganic arsenic [As(III) + As(V)] and 0.38% for DMAA. To ensure that the shelf life of the CRM has a validity period of two years, the ults for each analyte was also expanded to cover a period of two years by multiplying the standard error of the slope with the projected shelf life. Hence, final relative standard uncertainties for the analytes were 2.40% for cadmium, 2.76% for lead, 3.88% for total arsenic, 2.14% for inorganic arsenic [As(III) + As(V)] and 2.03% for DMAA.
Fig. 1. Long-term stability of cadmium (−), lead (+), total arsenic (), inorganic arsenic (•) and DMAA (x) content in apple juice.
3.3. Assignment of reference values Assignment of reference values were carried out on at least three bottles with two sub-samples taken from each bottle. The results were subjected to two outlier tests (Grubbs and Dixon’s) to identify the presence of outliers [14,28,29]. As there was no outlier in the data sets for all the analytes, the mean of results of the six subsamples were then taken as the reference values. For cadmium, the measured isotope ratios of the bracketing standard solution was compared with the ratio of the isotope abundances listed in the IUPAC table for mass bias correction [30]. Corrections were also done for isobaric interferences of molybdenum oxide (96 Mo16 O, 98 Mo16 O) and tin (112 Sn, 114 Sn) on 112 Cd and 114 Cd. The reference value for cadmium was calculated based on 114 Cd/111 Cd. However, the reference value based on 112 Cd/111 Cd was also calculated for confirmation and the difference in the results from the two isotope pairs was considered in the measurement uncertainty. The isotopic composition of lead had to be determined experimentally as it varies in nature. Hence, a standard solution prepared from SRM 981 (certified for lead isotopic composition) was analyzed before and after the sample for mass bias correction. Lead isotopes were measured (204 Pb, 206 Pb, 207 Pb and 208 Pb) together with 202 Hg to correct for isobaric interference of 204 Hg on 204 Pb. For lead, the reference value was calculated based on 208 Pb/206 Pb. A second pair of isotopes, 207 Pb/206 Pb, was used for confirmation. The difference in the results from the two isotope pairs was also considered in the measurement uncertainty. For determination of total arsenic content, the intensity ratio of 75 As/69 Ga was measured. In order to ensure optimum precision in the measurement of intensity ratios by ICP–HR–MS, the intensity ratio of 75 As/69 Ga was kept in between 0.1 to 10. More details on the method can be found in the publication by Ng et al. [22]. For determination of individual arsenic species in apple juice, the different arsenic species were separated through the HPLC column and measured by ICP–MS. The elution profiles (Fig. 2) shows that the different species could be well separated both in the sample and in the mixed standard solution. As(III) and As(V) were spiked into the samples and measured in the same sequence. The quantification was done for individual species and the results were added up together as inorganic arsenic species. DMAA was spiked and measured separately from the inorganic arsenic, to ensure that the DMAA and As(III) peaks did not overlap with each other. To estimate measurement uncertainty from characterization of lead and cadmium, the uncertainties from each component in the
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
5
Table 4 Summary of certified and measured value for brown rice (CRM 7532-a) and white rice flour (CRM 7503-a) CRMs. Analytes
Certified
Measured
Recovery (%)
Mass Fraction (mg/kg, as As)
Expanded uncertainty/ (mg/kg, as As), k = 2
Mass Fraction (mg/kg, as As)
Expanded uncertainty/ (mg/kg, as As), k = 2
CRM 7532-a As(III) + As(V) DMAA
0.298 0.0186
0.008 0.0008
0.296 0.0195
0.028 0.0015
99.3 104.8
CRM 7503-a As(III) + As(V) DMAA
0.0841 0.0133
0.0030 0.0009
0.0819 0.0133
0.0057 0.0015
97.4 100.0
Fig. 2. Elution profile of (a) As(III), As(V) and DMAA from apple juice sample and (b) As(III), As(V), MMA and DMAA from mixed standard (approximately 2.5 ppb each).
IDMS equations (Eqs. (1) and (2)) were combined according to the ISO Guide to the Expression of Uncertainty of Measurement (GUM) approach [31]. Similarly, the uncertainties from each component in the standard addition equations (Eqs. (3) and (4)) were estimated for total arsenic and arsenic species contents in apple juice. Any additional factors (MP and Fconf1 ) contributing to biases in the results were also considered. The combined uncertainty was expanded to 95% confidence interval using the coverage factor k = 2. CX = MP · Fconf 1 · CZ ·
AX MY × MZc RY − RSB RCB − RZ · · · · AZ MX × MYc RSB − RX RY − RCB
CX = MP · Fconf 1 · CZ ·
RiX RiZ
MY × MZc RY − RSB RCB − RZ · · MX × MYc RSB − RX RY − RCB
(1)
(2)
CX = MP · CZ ·
MD × MZ RU · MX × MS R S − R U
(3)
CX = MP · CS ·
MD × MF MX × MS
(4)
where MP is the factor representing any bias in precision due the sample inhomogeneity, weighing and ratio measurements (not applicable to Eq. (4)); Fconf1 is the factor representing any bias in the result value due to choice of ion pair; CX is the concentration of analyte in sample (mg/kg); CZ is the concentration of analyte in standard solution used to prepare calibration blend (for IDMS) or in calibration solution used for spiking (for standard addition) (mg/kg); CS is the concentration of analyte in unspiked sample after 2nd dilution (mg/kg); MX is the mass of sample digested or added to sample blend (g); MZC is the mass of standard solution added to calibration blend (g); MY is the mass of spike solution added to sample blend (g); MYC is the mass of spike solution added to calibration blend (g); MD is the total mass of digest after 1st dilution (g); MZ is the mass of calibration solution used (g); MS is the mass of diluted digest used to prepare unspiked and spiked solution (g); MF is the mass of final solution after 2nd dilution (g); AX is the relative atomic mass of analyte in sample; AZ is the relative atomic mass of
analyte in standard; RX is the isotopic amount ratio in sample; RY is the isotopic amount ratio in spike; RZ is the isotopic amount ratio in standard solution used to prepare calibration blend; RSB is the isotopic amount ratio in sample blend; RCB is the isotopic amount ratio in calibration blend; RiX is the sum of all isotope amount ratios of analyte in the sample; RiZ is the sum of all isotope amount ratios of analyte in the standard solution used to prepare calibration blend; R’U is the intensity ratio (analyte/internal standard) in unspiked solution and R’S is the intensity ratio (analyte/internal standard) in spiked solution. Table 5 summarizes the contributions of uncertainty from different factors. For cadmium, the major contributor in the overall uncertainty budgets was method precision (MP). While for lead, it was from the isotope ratios in sample, spike and standard solutions. For total arsenic and arsenic species, the major contributors in the overall uncertainty budgets were method precision (MP) and linear regression (CS ). The relative standard uncertainties from characterization (uchar ) were 0.46% for cadmium, 0.59% for lead, 0.55% for total arsenic, 4.07% for inorganic arsenic and 3.66% for DMAA. 3.4. Overall measurement uncertainty The combined uncertainties for the certified mass fractions were obtained from the combination of uncertainty from homogeneity (ubb ), long term stability (ults ) and characterization (uchar ) (Eq. (5)). uc 2 = ubb 2 + ults 2 + uchar 2
(5)
Table 6 summarizes the contribution of each factor to the overall combined uncertainties for the analytes in the apple juice. The major contribution to the overall combined uncertainties for cadmium, lead and total arsenic came from the uncertainty of long term stability (ults ), while the contribution from the uncertainty of homogeneity (ubb ) and characterization (uchar ) were minor. For the arsenic speciation, the main contribution came from the uncertainties of characterization (uchar ), while the contribution from the uncertainty of homogeneity (ubb ) and long term stability (ults ) were
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
6
Table 5 Uncertainty components from different factors contributing to the characterization of all the analytes in the apple juice CRM. Uncertainty component
Weighing
CS CZ RX , RY and RZ RiX and RiZ AX and AZ MP Fconf1
Contribution to uchar (%) Cadmium
Lead
Total Arsenic
Inorganic Arsenic
DMAA
MX (SB) 0.03%; MY (SB) 2.60%; MZ (CB) 0.03%; MY (CB) 2.54% – 9.77% 0.02% – – 75.20% 9.81%
MX (SB) 0.02%; MY (SB) 1.08%; MZ (CB) 0.02%; MY (CB) 0.98% – 1.80% 59.24% 8.09% <0.01% 17.99% 10.77%
MX 0.02%; MZ 1.58%; contribution from other weighings were below 0.01% – 2.00% – – – 96.39% –
<0.01%
<0.01%
53.07% – – – – 46.93% –
8.59% – – – – 91.41% –
Table 6 The contributions of uncertainty from characterization, homogeneity and long term stability and the certified mass fractions of all the analytes in the apple juice CRM. Uncertainty component
Cadmium
Lead
Total Arsenic
Inorganic Arsenic
DMAA
Relative standard uncertainty of between-bottle homogeneity (ubb , %) Relative standard uncertainty of long term stability (ults , %) Relative standard uncertainty of characterization (uchar , %) Combined relative standard uncertainty (uc , %) Combined standard uncertainty (uc , mg/kg) Certified concentration (mg/kg)
0.20 2.40 0.46 2.45 0.0054 0.220 ± 0.011
0.28 2.76 0.59 2.84 0.0070 0.245 ± 0.014
0.52 3.88 0.55 3.95 0.0073 0.185 ± 0.015
1.16 2.14 4.07 4.74 0.0059 0.124 ± 0.012
0.99 2.03 3.66 4.30 0.0026 0.0601 ± 0.0052
less significant. Overall, the combined relative standard uncertainties (uc ) were 2.45%, 2.84%, 3.95%, 4.74% and 4.30% for cadmium, lead, total arsenic, inorganic arsenic and DMAA, respectively. 4. Conclusions Exact matching IDMS and standard addition methods were developed for the certification of an apple juice material. The certified values are traceable to the International System of Units (SI). Since there is no available CRM for arsenic speciation in a fruit juice matrix, this material will be useful for routine testing laboratories in validating their methods or as a quality control material. This, in turn, will help to support existing regulations and uphold legislation for food safety. Acknowledgement The authors are grateful to the Health Sciences Authority, Singapore for the support of this project. References [1] WHO, Guidelines for Drinking-Water Quality; Guidelines, Vol. 1, 3rd ed. (pp. 145–196 g), 2008. http://www.who.int/water sanitation health/dwq/gdwq3/ en/ (Accessed 8 June 2016). [2] FDA, Beverages Bottled Water; Code of Federal Regulations. 21 CFR 165.110., 2007. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch. cfm/ (accessed 8 June 2016). [3] EPA, 2011 Edition of the Drinking Water Standards and Health Advisories, EPA, Office of Water, Washington, DC, 2011. http://www.epa.gov/ waterscience/criteria/drinking/dwstandards.pdf (Accessed 8 June 2016). [4] FDA, Guidance for Industry: Juice HACCP Hazard and Controls Guidance, 2004, http://www.fda.gov/Food/GuidanceRegulation/ GuidanceDocumentsRegulatoryInformation/Juice/ucm072557.htm (accessed 11 May 2016). [5] Codex Alimentarius, Maximum Levels of Lead in Fruit, Juices and Canned Foods, Codex Alimentarius, International Food Standards, 2015, http://ftp.fao.org/codex/reports/reports 2015/REP15 CFe.pdf (Accessed 2 August 2016). [6] FDA, FDA Proposes “Action Level” for Arsenic in Apple Juice: Agency Testing and Analysis Confirm Overall Safety of Apple Juice, 2013, http://www.fda.gov/ NewsEvents/Newsroom/PressAnnouncements/ucm360466.htm (Accessed 21 December 2015). [7] AVA, Sale of Food Act; Chapter 283, Section 56 (1) Food Regulations, 2006, http://www.ava.gov.sg/docs/default-source/legislation/sale-of-food-act/2web sof food-regulations-15-dec-2014.pdf (Accessed 21 December 2015).
[8] S. Satarug, M.R. Moore, Adverse health effects of chronic exposure to low-level cadmium in foodstuffs and cigarette smoke, Environ. Med. 112 (2004) 1099–1103. [9] P.A. Meyer, M.J. Brown, H. Falk, Global approach to reducing lead exposure and poisoning, Mutat. Res. 659 (2008) 166–175. [10] L. Järup, Hazards of heavy metal contamination, Br. Med. Bull. 68 (2003) 167–182. [11] B.K. Mandal, K.T. Suzuki, Arsenic round the world: a review, Talanta 58 (2002) 201–235. [12] B.E. Hettick, J.E. Can˜as-Carrell, A.D. French, D.M. Klein, Arsenic: a review of the element˜s toxicity, plant interactions: and potential methods of remediation, J. Agric. Food Chem. 63 (2015) 7097–7107. [13] FDA, Guidance for Industry Arsenic in Apple Juice: Action Level, 2015, http:// www.fda.gov/downloads/Food/GuidanceRegulation/ GuidanceDocumentsRegulatoryInformation/ ChemicalContaminantsMetalsNaturalToxinsPesticides/UCM360048.pdf (Accessed 21 December 2015). [14] International Standards Organization, ISO Guide 34, General Requirements for the Competence of Reference Material Producers, ISO, Switzerland, 2009. [15] International Standards Organization, ISO Guide 35, Reference Materials–General and Statistical Principles for Certification, ISO, Switzerland, 2006. [16] D. Tolen, ILAC Discussion Paper on Homogeneity and Stability Testing, 2008, https://www.gso.org.sa/gso-website/gso-website/activities-ar/accreditation/ pt/ptcg-hands-april0808.pdf (Accessed 2 August 2016). [17] L.G. Mackay, C.P. Taylor, R.B. Myors, R. Hearn, B. King, High accuracy analysis by isotope dilution mass spectrometry using iterative exact matching technique, Accredit. Qual. Assur. 8 (2003) 191–194. [18] A. Henrion, Reduction of systematic errors in quantitative analysis by isotope dilution mass spectrometry (IDMS): an iterative method, Fresenius J. Anal. Chem. 350 (1994) 657–658. [19] Y. Zhu, K. Chiba, Internal standard method coupled with a gravimetric standard addition method for elemental measurement by ICP-MS, J. Anat. At. Spectrom. 27 (2012) 1000–1006. [20] P. Abbyad, J. Tromp, J. Lam, E. Salin, Optimisation of the technique of standard additions for inductively coupled plasma mass spectrometry, J. Anal. At. Spectrom. 16 (2001) 464–469. [21] S.Y. Ng, Z. Aimei, L.P. Sim, Y. Ding, K.L. Yuen, R.Y.C. Shin, T.K. Lee, High accuracy measurements of trace elements in drinking water by exact matching isotope dilution mass spectrometry (IDMS), Int. J. Mass Spectrom. 321–322 (2012) 19–24. [22] S.Y. Ng, F. Dewi, J. Wang, L.P. Sim, R.Y.C. Shin, T.K. Lee, Development of a cosmetic cream certified reference material: certification of lead: mercury and arsenic mass fractions in cosmetic cream, Int. J. Mass Spectrom. 389 (2015) 59–65. [23] G. Chew, L.P. Sim, S.Y. Ng, Y. Ding, R.Y.C. Shin, T.K. Lee, Development of a mushroom powder certified reference material for calcium, arsenic: cadmium and lead measurements, Food Chem. 190 (2016) 293–299. [24] T. Narukawa, K. Chiba, Heat-assisted aqueous extraction of rice flour for arsenic speciation analysis, J. Agric. Food Chem. 58 (2010) 8183–8188. [25] T. Narukawa, A. Hioki, K. Chiba, Speciation and monitoring test for inorganic arsenic in white rice flour, J. Agric. Food Chem. 60 (2012) 1122–1127.
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model MASPEC-15703; No. of Pages 7
ARTICLE IN PRESS F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
[26] T. Narukawa, T. Suzuki, K. Inagaki, A. Hioki, Extraction techniques for arsenic species in rice flour and their speciation by HPLC-ICP-MS, Talanta 130 (2014) 213–220. [27] J.-M. Mermet, Limit of quantification in atomic spectrometry: an ambiguous concept? Spectrochim. Acta B 63 (2008) 166–182. [28] F.E. Grubbs, Procedures for detecting outlying observations in samples, Technometrics 11 (1969) 1–21.
7
[29] R.B. Dean, W.J. Dixon, Simplified statistics for small numbers of observations, Anal. Chem. 23 (1951) 636–638. [30] M. Berglund, M.E. Wieser, Isotopic compositions of the elements 2009 (IUPAC Technical Report), Pure Appl. Chem. 83 (2011) 397–410. [31] Joint Committee for Guides in Metrology, JCGM 100:2008 Evaluation of Measurement Data – Guide to the Expression of Uncertainty in Measurement, JCGM, France, 2008.
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
International Journal of Mass Spectrometry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Young Scientist Feature
Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species Fransiska Dewi, Lay Peng Sim, Juan Wang, Benny M.K. Tong, Richard Y.C. Shin ∗ , Tong Kooi Lee Chemical Metrology Laboratory, Chemical Metrology Division, Applied Sciences Group, Health Sciences Authority of Singapore, 1 Science Park Road, #01-05/06, The Capricorn, Singapore Science Park II, Singapore 117528, Singapore
a r t i c l e
i n f o
Article history: Received 20 September 2016 Received in revised form 1 November 2016 Accepted 2 November 2016 Available online xxx Keywords: Certified reference material Apple juice Toxic elements Arsenic speciation Isotope dilution mass spectrometry Standard addition
a b s t r a c t In the development of the apple juice certified reference material, isotope dilution mass spectrometry method was used for the assignment of certified values and long-term stability study for cadmium and lead. For total arsenic, inorganic arsenic, as well as dimethylarsenic acid, standard addition method was used. The analytes were found to be homogenous and stable over a period of at least 4.5 months at storage temperature of −20 ◦ C (long-term stability). The certified mass fraction values were (0.220 ± 0.011) mg/kg for cadmium, (0.245 ± 0.014) mg/kg for lead, (0.185 ± 0.015) mg/kg for total arsenic, (0.124 ± 0.012) mg/kg for inorganic arsenic [As(III)+As(V)] and (0.0601 ± 0.0052) mg/kg for DMAA. The recovery of the arsenic species was over 99%. This apple juice certified reference material can be used for method validation or as a quality control material by routine testing laboratories. © 2016 Elsevier B.V. All rights reserved.
1. Introduction For drinking water, the maximum contaminant level for cadmium has been set at 3 g/L according to the World Health Organization (WHO) and 5 g/L according to the US Food and Drug Administration (FDA) and the Environmental Protection Agency (EPA) [1–3]. FDA has also established a guidance level of 50 ppb of lead in ready-to-drink fruit juices, in line with the Codex Alimentarius Commission standard [4,5]. In addition, FDA has proposed an “action level” for inorganic arsenic in apple juice to be 10 g/L [6]. It is the same level as for arsenic in drinking water set by the EPA [3]. Meanwhile, the Agri-Food and Veterinary Authority of Singapore (AVA) has regulated the maximum amount of lead and arsenic in fruit juices to be 300 and 200 ppb, respectively [7]. There is no limit specified for cadmium in fruit juices. However, the Sale of Food Act mentions that natural mineral water and any article of food shall not contain more than 10 ppb and 200 ppb of cadmium, respectively [7]. Cadmium is considered a cumulative nephrotoxicant, whose level in human organs increases with age due to the lack of an active biochemical process for its elimination coupled with renal
∗ Corresponding author. E-mail address: Richard [email protected] (R.Y.C. Shin).
reabsorption. There has been accumulating evidence for the carcinogenic risk of chronic cadmium exposure [8]. Lead, on the other hand, has the ability to affect almost every system in the human body, including the reproductive, neurological, hematopoietic, hepatic and renal systems [9]. Long term low level exposure in children is harmful for the brain and nervous system [10]. As in cadmium and lead, acute and chronic exposures to arsenic can also cause adverse health effects to human including dermal changes, damage to internal organs and carcinogenic effects [11,12]. Arsenic found in fruits and vegetables is primarily organic arsenic and only less than 10% of the arsenic is present in the inorganic form [11]. Inorganic arsenic in apple juice may come from processing aids, prior and/or current use of pesticides containing arsenic on apple orchards, natural arsenic in soil or water and atmospheric deposition from industrial activities [13]. In human, organoarsenic compounds including monomethylarsinic acid (MMAA) and dimethylarsinic acid (DMAA) are not readily taken up by the cell, subjected to limited metabolism and excreted close to their original form in the urine [11]. In contrast, the soluble inorganic arsenic can be absorbed and accumulated in tissues and body fluids. Hence, inorganic arsenic species are more toxic than the organic ones and As(III) is more toxic than As(V) [11,12]. Arsenic speciation gives valuable information that helps in judging the actual level of toxicity.
http://dx.doi.org/10.1016/j.ijms.2016.11.001 1387-3806/© 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
2
This paper describes the development of an apple juice certified reference material (CRM) containing cadmium, lead, inorganic [As(III) and As(V)] as well as organoarsenic (DMAA) species. The development of the CRM, including procedures for sample preparation, homogeneity study, stability study, assignment of reference values and uncertainty evaluation, was carried out in accordance with the requirements of ISO Guides 34 and 35 [14,15]. Prior to establishing the certified mass fraction values for the analytes in the material, a method development was conducted using CRMs from the National Metrology Institute of Japan (NMIJ) (Tsukuba, Japan). The results obtained were in good agreement with certified concentration levels. To the best of our knowledge, there are currently no available CRMs for arsenic speciation in fruit juice matrix. Hence, rice flour CRMs were used in the method development. 2. Material and methods 2.1. Reagents, standard solutions and CRMs All experimental work was carried out in class 100 low laminar flow fumehoods or a class 10,000 clean room. Standard and sample solutions were prepared gravimetrically on a microanalytical balance (Mettler Toledo XP205 or XP26, Greifensee, Switzerland). The certified reference standards for cadmium, lead and total arsenic were obtained from the National Institute of Standards and Technology (NIST) (Gaithersburg, MD, USA). Isotopically enriched isotopes were purchased from Oak Ridge National Laboratory (Oak Ridge, TN, USA). For determination of cadmium, lead and total arsenic, all working solutions were diluted with 5% HNO3 (aq). Nitric acid (67–70% HNO3 , Ultrapur-100, Kanto Kagaku Singapore Pte Ltd) used was distilled twice using DST-1000 sub-boiling distillation system (Savillex Corporation, MN, USA). The As(III), As(V) and DMAA standards were obtained from the National Metrology Institute of Japan (NMIJ) (Tsukuba, Japan). Stock solution for As(III) was prepared by dissolving As(III) trioxide powder in 1% NaOH (aq), while stock solution for As(V) and DMAA were prepared by diluting with Milli-Q element water (18.2 M cm, Millipore Corporation, MA, USA). The certified reference standards and isotopically enriched isotopes used are listed in Table 1. The brown rice (CRM 7532-a) and white rice (CRM 7503-a) CRMs used for method development were obtained from NMIJ. The high-performance liquid chromatography (HPLC) buffer solution used for arsenic speciation was prepared by adding 0.7 g tetramethylammonium hydroxide pentahydrate (99%, Aik Moh Paints and Chemicals, Singapore), 1.6 g sodium 1-butanesulfonate (≥ 99.0%, Sigma Aldrich, MO, USA), 0.7 g malonic acid (Reagentplus 99%, Sigma Aldrich, MO, USA) and 0.5 mL methanol (HPLC grade, J.T. Baker, PA, USA) into 1 L of Milli-Q water. The pH of the buffer solution was approximately 3.0. 2.2. Instrumentations An Agilent Technologies 7700x ICP–MS system (ICP–MS) (Agilent Technologies International Japan, Ltd., Tokyo, Japan) equipped with an octopole collision cell was used for measurements of cadmium and lead. Measurements of total arsenic were carried out using a Finnigan Element 2 (Finnigan MAT GmbH, Bremen, Germany) sector-field ICP–MS (SF–ICP–MS) using a self-aspirating PFA MicroFlow nebulizer. For arsenic speciation, an Agilent Technologies 1200 HPLC was coupled with the ICP–MS system to separate and measure the different arsenic species [As(III), As(V) and DMAA]. The HPLC was equipped with an automatic sample injector for direct sample introduction into the ICP–MS. The instruments were conditioned and optimized daily to achieve optimum
sensitivity and stability. The typical operating conditions and data acquisition parameters are summarized in Table 2. 2.3. Preparation of candidate CRM sample The apple juice material, obtained from a local supermarket, was screened and found to contain negligible amount of cadmium, lead and arsenic. The material was filtered using Nalgene RapidFlowTM Filter Units (90 mm nylon membrane, 0.2 m pore, VWR, PA, USA), preserved with sodium benzoate (1.2 g/L, >98.0%, HPLC grade, Tokyo Chemical Industry, Tokyo, Japan) and spiked with cadmium nitrate tetrahydrate, lead (II) nitrate from Sigma Aldrich (MO, USA) as well as As(III) trioxide, As(V) and DMAA from NMIJ. The mixture was then homogenized by mixing on a shaker for 1 h and bottled into 125-mL high-density polyethylene (HDPE) bottles. Each bottle containing 100 mL of apple juice was flushed with nitrogen gas for 15 s before sealing and labelled according to its dispensing sequence number. A total of fifty four bottles were prepared. The targeted ranges of concentration for cadmium, lead, inorganic arsenic and DMAA in the apple juice were 0.2–0.4 mg/kg, 0.2–0.4 mg/kg, 0.02–0.2 mg/kg and 0.01–0.1 mg/kg, respectively. The apple juice was not acidified so that it would closely match in terms of matrix, measurands and concentrations to the type of materials encountered in routine testing or calibration. For long term storage, the material was stored in frozen state at −20 ◦ C to reduce the risk of deterioration. 2.4. Homogeneity study, stability study and assignment of reference values The homogeneity of the material was established by selecting six bottles using a stratified random sampling scheme. The samples were analyzed by ICP–MS using isotope dilution mass spectrometry (IDMS) for cadmium and lead, by SF–ICP–MS using standard addition method for total arsenic and by HPLC–ICP–MS using external calibration method for arsenic species. The homogeneity of each analytes was tested using the one-way analysis of variance (ANOVA) on duplicate results from these six bottles. The result from the homogeneity study of total arsenic was also used for assignment of reference value. For the assignment of reference values three bottles were randomly selected and two subsamples were taken from each bottle. The measurements were carried out using IDMS (cadmium and lead) and standard addition (arsenic species) methods. The results were taken as the first point (t = 0 month) in the long-term stability study. Two more stability points were determined at time interval of 3 and 4.5 months, using the classical design [16]. 2.5. Sample preparation for analysis For determination of cadmium, lead and total arsenic content, a multiple spiking approach was used for the sample preparation. Calculated amounts of enriched isotope spike solutions (111 Cd and 206 Pb) were added gravimetrically into approximately 1 g of apple juice. The mixture was digested with 2.5 mL of concentrated HNO3 at room temperature overnight. Afterwards, a calculated amount of gallium (Ga) standard solution (SRM 3119a, NIST, Gaithersburg, MD, USA) was added into the digest and the mixture was diluted to 50 g using Type I Milli-Q water. The final weight of the diluted digest was recorded. The digest was then used to prepare sample blends (cadmium and lead) as well as unspiked and spiked solutions (total arsenic). This protocol was employed for the homogeneity testing, assignment of reference values as well as long-term stability studies. For arsenic speciation, approximately 0.5 g (for external calibration) or 1 g (for standard addition) of apple juice was diluted with
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
3
Table 1 Certified reference standards used. Analytes
Certified Reference Standard
Cadmium Lead Arsenic Gallium As(III) As(V) DMAA a b
Isotope Spike (Isotopic Fraction)
Code
Certified Value
SRM 3108 SRM 3128 SRM 981a SRM 3103a SRM 3119ab CRM 3003-a CRM 7912-a CRM 7913-a
(10.007 ± 0.027) mg/g (9.995 ± 0.014) mg/g – (9.999 ± 0.015) mg/g (10.00 ± 0.04) mg/g (100.001 ± 0.018) mg/kg (99.53 ± 1.67) mg/kg (25.11 ± 0.70) mg/kg
111 206
Cd (96.44%) Pb (99.76%)
– – – – – –
Used for mass bias correction. Used as an internal standard.
Table 2 Operating conditions for ICP–MS, SF–ICP–MS and HPLC instruments. ICP-MS (Agilent 7700x) Element
Cadmium
RF power/W Carrier gas flow rate/L min−1 Make up gas flow rate/L min−1 Sampling depth/mm Spray chamber temperature/◦ C Ion lens settling Helium gas flow/mL min−1 Data acquisition integration time/s Points per peak Repetitions m/z
4.7
97 (Mo); 111, 112, 114 (Cd); 118 (Sn)
Lead 1550 0.9 0.3 8 2 Optimized daily 4.7 3 3 3 202 (Hg); 204, 206, 207, 208 (Pb)
Arsenic (speciation)
3.0
35 (Cl); 75 (As)
SF-ICP-MS (Element 2) Element
Arsenic
RF power/W Scanning mode Settling time/ms Resolution Sample time/ms Samples per peak Mass window Runs Passes Total time per sample/min m/z
1250 E-scan 1 High (10,000) 100 20 100 5 6 2.01 69, 71 (Ga); 75 (As)
Agilent 1200 HPLC Element
Arsenic (speciation)
Column Mobile phase Temperature/◦ C Flow rate/mL min-1 Total elution time/min
L-ODS or C18 MG-S5 (4.6 ID x 150 mm, 5 m, Waters type, Chemical Evaluation and Research Institute, Japan) 10 mM sodium 1-butanesulfonate/4 mM malonic acid/4 mM tetramethylammonium hydroxide/0.05% methanol in type I water (pH 3.0) 30 0.5 6
Type I Milli-Q water to approximately 10 g. For homogeneity study using external calibration, no further dilution was required before sample analysis. For the assignment of reference values and longterm stability studies using standard addition, the diluted sample was used to prepare unspiked and spiked solutions. The samples were filtered through a 0.2 m filter (regenerated cellulose, 15 mm, Agilent, CA, USA) and transferred into crimp snap polypropylene vials (Agilent, CA, USA) for analysis using HPLC–ICP–MS.
2.6. Analytical methods—exact-matching IDMS, standard addition multi-point calibration and standard addition by intensity ratio
spikes were added into the sample and standard solutions. Total arsenic content in apple juice was determined using standard addition method by measuring intensity ratio of arsenic to gallium (internal standard) [19]. An unspiked solution and four spiked solutions were prepared from each diluted digest. The analysis of arsenic species in apple juice was performed using multi-point calibration standard addition technique [20]. Different amounts of arsenic species standard solution were spiked into the sample to produce an unspiked and four spiked solutions. The details of the methods can be found in our earlier publications [21–23]. At least two reagent blanks were also prepared by subjecting them to all sample preparation steps to evaluate possible blank contributions. All measured intensity ratios were subjected to blank correction before the concentration of each analyte was calculated.
Exact-matching IDMS method [17,18] was employed in the measurement of cadmium and lead. Known amounts of isotopic Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
4 Table 3 LODs and LOQs of As(III), As(V) and DMAA. Analytes
LOD (mg/kg)
LOQ (mg/kg)
As(III) As(v) DMAA
0.00041 0.00017 0.00029
0.00137 0.00056 0.00096
3. Results and discussion 3.1. Method development using brown rice (CRM 7532-a) and white rice flour (CRM 7503-a) Since there are no commercially available fruit juice CRM for arsenic speciation, rice flour CRMs (CRM 7532-a and CRM 7503a) were used for the method development. The different type of arsenic species [As(III), As(V) and DMAA] are also commonly found in rice flour. Unlike apple juice, arsenic speciation in rice flour requires an extraction procedure. Water extraction (for CRM 7503a) or acid extraction using diluted HNO3 (1% v/v) (for CRM 7532-a) was carried out using microwave-assisted digestion method (95 ◦ C for 90 min) [24–26]. The mass fractions of As(III), As(V) and DMAA were determined by using multi-point calibration standard addition method. The arsenic species were separated by HPLC and subsequently measured by ICP-MS. Table 3 shows the limit of detections (LODs) and the limit of quantifications (LOQs) of the method for the different species [27]. Since As(III) and As(V) can undergo inter-conversion during extraction, the mass fraction of the total inorganic arsenic [As(III) + As(V)] are usually reported, instead of the individual species. Good agreement between the measured and certified values was observed for both inorganic arsenic and DMAA (see Table 4). Hence, the developed method was deemed suitable and was adopted for the quantification of the arsenic species in apple juice.
3.2. Homogeneity and stability studies Homogeneity testing was performed on six bottles with two sub-samples taken from each bottle. No significant differences in the between and within-bottle variances were found using oneway ANOVA at 95% confidence level [14]. Thus, the material was regarded to be sufficiently homogeneous. The relative standard uncertainty due to between-bottle inhomogeneity (ubb ) were 0.20% for cadmium, 0.28% for lead, 0.52% for total arsenic, 1.16% for inorganic arsenic [As(III) + As(V)] and 0.99% for DMAA. The long-term stability of the analytes in apple juice was assessed over time interval of 3 and 4.5 months. Two subsamples from two bottles of the material were analyzed at each time point. The analytes in apple juice were shown to be stable over a period of at least 4.5 months (see Fig. 1). The relative standard uncertainties due to long-term storage (4.5 months or approximately 20 weeks) of the material (ults ) estimated from the standard error of the slope were 0.47% for cadmium, 0.55% for lead, 0.76% for total arsenic, 0.40% for inorganic arsenic [As(III) + As(V)] and 0.38% for DMAA. To ensure that the shelf life of the CRM has a validity period of two years, the ults for each analyte was also expanded to cover a period of two years by multiplying the standard error of the slope with the projected shelf life. Hence, final relative standard uncertainties for the analytes were 2.40% for cadmium, 2.76% for lead, 3.88% for total arsenic, 2.14% for inorganic arsenic [As(III) + As(V)] and 2.03% for DMAA.
Fig. 1. Long-term stability of cadmium (−), lead (+), total arsenic (), inorganic arsenic (•) and DMAA (x) content in apple juice.
3.3. Assignment of reference values Assignment of reference values were carried out on at least three bottles with two sub-samples taken from each bottle. The results were subjected to two outlier tests (Grubbs and Dixon’s) to identify the presence of outliers [14,28,29]. As there was no outlier in the data sets for all the analytes, the mean of results of the six subsamples were then taken as the reference values. For cadmium, the measured isotope ratios of the bracketing standard solution was compared with the ratio of the isotope abundances listed in the IUPAC table for mass bias correction [30]. Corrections were also done for isobaric interferences of molybdenum oxide (96 Mo16 O, 98 Mo16 O) and tin (112 Sn, 114 Sn) on 112 Cd and 114 Cd. The reference value for cadmium was calculated based on 114 Cd/111 Cd. However, the reference value based on 112 Cd/111 Cd was also calculated for confirmation and the difference in the results from the two isotope pairs was considered in the measurement uncertainty. The isotopic composition of lead had to be determined experimentally as it varies in nature. Hence, a standard solution prepared from SRM 981 (certified for lead isotopic composition) was analyzed before and after the sample for mass bias correction. Lead isotopes were measured (204 Pb, 206 Pb, 207 Pb and 208 Pb) together with 202 Hg to correct for isobaric interference of 204 Hg on 204 Pb. For lead, the reference value was calculated based on 208 Pb/206 Pb. A second pair of isotopes, 207 Pb/206 Pb, was used for confirmation. The difference in the results from the two isotope pairs was also considered in the measurement uncertainty. For determination of total arsenic content, the intensity ratio of 75 As/69 Ga was measured. In order to ensure optimum precision in the measurement of intensity ratios by ICP–HR–MS, the intensity ratio of 75 As/69 Ga was kept in between 0.1 to 10. More details on the method can be found in the publication by Ng et al. [22]. For determination of individual arsenic species in apple juice, the different arsenic species were separated through the HPLC column and measured by ICP–MS. The elution profiles (Fig. 2) shows that the different species could be well separated both in the sample and in the mixed standard solution. As(III) and As(V) were spiked into the samples and measured in the same sequence. The quantification was done for individual species and the results were added up together as inorganic arsenic species. DMAA was spiked and measured separately from the inorganic arsenic, to ensure that the DMAA and As(III) peaks did not overlap with each other. To estimate measurement uncertainty from characterization of lead and cadmium, the uncertainties from each component in the
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
5
Table 4 Summary of certified and measured value for brown rice (CRM 7532-a) and white rice flour (CRM 7503-a) CRMs. Analytes
Certified
Measured
Recovery (%)
Mass Fraction (mg/kg, as As)
Expanded uncertainty/ (mg/kg, as As), k = 2
Mass Fraction (mg/kg, as As)
Expanded uncertainty/ (mg/kg, as As), k = 2
CRM 7532-a As(III) + As(V) DMAA
0.298 0.0186
0.008 0.0008
0.296 0.0195
0.028 0.0015
99.3 104.8
CRM 7503-a As(III) + As(V) DMAA
0.0841 0.0133
0.0030 0.0009
0.0819 0.0133
0.0057 0.0015
97.4 100.0
Fig. 2. Elution profile of (a) As(III), As(V) and DMAA from apple juice sample and (b) As(III), As(V), MMA and DMAA from mixed standard (approximately 2.5 ppb each).
IDMS equations (Eqs. (1) and (2)) were combined according to the ISO Guide to the Expression of Uncertainty of Measurement (GUM) approach [31]. Similarly, the uncertainties from each component in the standard addition equations (Eqs. (3) and (4)) were estimated for total arsenic and arsenic species contents in apple juice. Any additional factors (MP and Fconf1 ) contributing to biases in the results were also considered. The combined uncertainty was expanded to 95% confidence interval using the coverage factor k = 2. CX = MP · Fconf 1 · CZ ·
AX MY × MZc RY − RSB RCB − RZ · · · · AZ MX × MYc RSB − RX RY − RCB
CX = MP · Fconf 1 · CZ ·
RiX RiZ
MY × MZc RY − RSB RCB − RZ · · MX × MYc RSB − RX RY − RCB
(1)
(2)
CX = MP · CZ ·
MD × MZ RU · MX × MS R S − R U
(3)
CX = MP · CS ·
MD × MF MX × MS
(4)
where MP is the factor representing any bias in precision due the sample inhomogeneity, weighing and ratio measurements (not applicable to Eq. (4)); Fconf1 is the factor representing any bias in the result value due to choice of ion pair; CX is the concentration of analyte in sample (mg/kg); CZ is the concentration of analyte in standard solution used to prepare calibration blend (for IDMS) or in calibration solution used for spiking (for standard addition) (mg/kg); CS is the concentration of analyte in unspiked sample after 2nd dilution (mg/kg); MX is the mass of sample digested or added to sample blend (g); MZC is the mass of standard solution added to calibration blend (g); MY is the mass of spike solution added to sample blend (g); MYC is the mass of spike solution added to calibration blend (g); MD is the total mass of digest after 1st dilution (g); MZ is the mass of calibration solution used (g); MS is the mass of diluted digest used to prepare unspiked and spiked solution (g); MF is the mass of final solution after 2nd dilution (g); AX is the relative atomic mass of analyte in sample; AZ is the relative atomic mass of
analyte in standard; RX is the isotopic amount ratio in sample; RY is the isotopic amount ratio in spike; RZ is the isotopic amount ratio in standard solution used to prepare calibration blend; RSB is the isotopic amount ratio in sample blend; RCB is the isotopic amount ratio in calibration blend; RiX is the sum of all isotope amount ratios of analyte in the sample; RiZ is the sum of all isotope amount ratios of analyte in the standard solution used to prepare calibration blend; R’U is the intensity ratio (analyte/internal standard) in unspiked solution and R’S is the intensity ratio (analyte/internal standard) in spiked solution. Table 5 summarizes the contributions of uncertainty from different factors. For cadmium, the major contributor in the overall uncertainty budgets was method precision (MP). While for lead, it was from the isotope ratios in sample, spike and standard solutions. For total arsenic and arsenic species, the major contributors in the overall uncertainty budgets were method precision (MP) and linear regression (CS ). The relative standard uncertainties from characterization (uchar ) were 0.46% for cadmium, 0.59% for lead, 0.55% for total arsenic, 4.07% for inorganic arsenic and 3.66% for DMAA. 3.4. Overall measurement uncertainty The combined uncertainties for the certified mass fractions were obtained from the combination of uncertainty from homogeneity (ubb ), long term stability (ults ) and characterization (uchar ) (Eq. (5)). uc 2 = ubb 2 + ults 2 + uchar 2
(5)
Table 6 summarizes the contribution of each factor to the overall combined uncertainties for the analytes in the apple juice. The major contribution to the overall combined uncertainties for cadmium, lead and total arsenic came from the uncertainty of long term stability (ults ), while the contribution from the uncertainty of homogeneity (ubb ) and characterization (uchar ) were minor. For the arsenic speciation, the main contribution came from the uncertainties of characterization (uchar ), while the contribution from the uncertainty of homogeneity (ubb ) and long term stability (ults ) were
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model
ARTICLE IN PRESS
MASPEC-15703; No. of Pages 7
F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
6
Table 5 Uncertainty components from different factors contributing to the characterization of all the analytes in the apple juice CRM. Uncertainty component
Weighing
CS CZ RX , RY and RZ RiX and RiZ AX and AZ MP Fconf1
Contribution to uchar (%) Cadmium
Lead
Total Arsenic
Inorganic Arsenic
DMAA
MX (SB) 0.03%; MY (SB) 2.60%; MZ (CB) 0.03%; MY (CB) 2.54% – 9.77% 0.02% – – 75.20% 9.81%
MX (SB) 0.02%; MY (SB) 1.08%; MZ (CB) 0.02%; MY (CB) 0.98% – 1.80% 59.24% 8.09% <0.01% 17.99% 10.77%
MX 0.02%; MZ 1.58%; contribution from other weighings were below 0.01% – 2.00% – – – 96.39% –
<0.01%
<0.01%
53.07% – – – – 46.93% –
8.59% – – – – 91.41% –
Table 6 The contributions of uncertainty from characterization, homogeneity and long term stability and the certified mass fractions of all the analytes in the apple juice CRM. Uncertainty component
Cadmium
Lead
Total Arsenic
Inorganic Arsenic
DMAA
Relative standard uncertainty of between-bottle homogeneity (ubb , %) Relative standard uncertainty of long term stability (ults , %) Relative standard uncertainty of characterization (uchar , %) Combined relative standard uncertainty (uc , %) Combined standard uncertainty (uc , mg/kg) Certified concentration (mg/kg)
0.20 2.40 0.46 2.45 0.0054 0.220 ± 0.011
0.28 2.76 0.59 2.84 0.0070 0.245 ± 0.014
0.52 3.88 0.55 3.95 0.0073 0.185 ± 0.015
1.16 2.14 4.07 4.74 0.0059 0.124 ± 0.012
0.99 2.03 3.66 4.30 0.0026 0.0601 ± 0.0052
less significant. Overall, the combined relative standard uncertainties (uc ) were 2.45%, 2.84%, 3.95%, 4.74% and 4.30% for cadmium, lead, total arsenic, inorganic arsenic and DMAA, respectively. 4. Conclusions Exact matching IDMS and standard addition methods were developed for the certification of an apple juice material. The certified values are traceable to the International System of Units (SI). Since there is no available CRM for arsenic speciation in a fruit juice matrix, this material will be useful for routine testing laboratories in validating their methods or as a quality control material. This, in turn, will help to support existing regulations and uphold legislation for food safety. Acknowledgement The authors are grateful to the Health Sciences Authority, Singapore for the support of this project. References [1] WHO, Guidelines for Drinking-Water Quality; Guidelines, Vol. 1, 3rd ed. (pp. 145–196 g), 2008. http://www.who.int/water sanitation health/dwq/gdwq3/ en/ (Accessed 8 June 2016). [2] FDA, Beverages Bottled Water; Code of Federal Regulations. 21 CFR 165.110., 2007. http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch. cfm/ (accessed 8 June 2016). [3] EPA, 2011 Edition of the Drinking Water Standards and Health Advisories, EPA, Office of Water, Washington, DC, 2011. http://www.epa.gov/ waterscience/criteria/drinking/dwstandards.pdf (Accessed 8 June 2016). [4] FDA, Guidance for Industry: Juice HACCP Hazard and Controls Guidance, 2004, http://www.fda.gov/Food/GuidanceRegulation/ GuidanceDocumentsRegulatoryInformation/Juice/ucm072557.htm (accessed 11 May 2016). [5] Codex Alimentarius, Maximum Levels of Lead in Fruit, Juices and Canned Foods, Codex Alimentarius, International Food Standards, 2015, http://ftp.fao.org/codex/reports/reports 2015/REP15 CFe.pdf (Accessed 2 August 2016). [6] FDA, FDA Proposes “Action Level” for Arsenic in Apple Juice: Agency Testing and Analysis Confirm Overall Safety of Apple Juice, 2013, http://www.fda.gov/ NewsEvents/Newsroom/PressAnnouncements/ucm360466.htm (Accessed 21 December 2015). [7] AVA, Sale of Food Act; Chapter 283, Section 56 (1) Food Regulations, 2006, http://www.ava.gov.sg/docs/default-source/legislation/sale-of-food-act/2web sof food-regulations-15-dec-2014.pdf (Accessed 21 December 2015).
[8] S. Satarug, M.R. Moore, Adverse health effects of chronic exposure to low-level cadmium in foodstuffs and cigarette smoke, Environ. Med. 112 (2004) 1099–1103. [9] P.A. Meyer, M.J. Brown, H. Falk, Global approach to reducing lead exposure and poisoning, Mutat. Res. 659 (2008) 166–175. [10] L. Järup, Hazards of heavy metal contamination, Br. Med. Bull. 68 (2003) 167–182. [11] B.K. Mandal, K.T. Suzuki, Arsenic round the world: a review, Talanta 58 (2002) 201–235. [12] B.E. Hettick, J.E. Can˜as-Carrell, A.D. French, D.M. Klein, Arsenic: a review of the element˜s toxicity, plant interactions: and potential methods of remediation, J. Agric. Food Chem. 63 (2015) 7097–7107. [13] FDA, Guidance for Industry Arsenic in Apple Juice: Action Level, 2015, http:// www.fda.gov/downloads/Food/GuidanceRegulation/ GuidanceDocumentsRegulatoryInformation/ ChemicalContaminantsMetalsNaturalToxinsPesticides/UCM360048.pdf (Accessed 21 December 2015). [14] International Standards Organization, ISO Guide 34, General Requirements for the Competence of Reference Material Producers, ISO, Switzerland, 2009. [15] International Standards Organization, ISO Guide 35, Reference Materials–General and Statistical Principles for Certification, ISO, Switzerland, 2006. [16] D. Tolen, ILAC Discussion Paper on Homogeneity and Stability Testing, 2008, https://www.gso.org.sa/gso-website/gso-website/activities-ar/accreditation/ pt/ptcg-hands-april0808.pdf (Accessed 2 August 2016). [17] L.G. Mackay, C.P. Taylor, R.B. Myors, R. Hearn, B. King, High accuracy analysis by isotope dilution mass spectrometry using iterative exact matching technique, Accredit. Qual. Assur. 8 (2003) 191–194. [18] A. Henrion, Reduction of systematic errors in quantitative analysis by isotope dilution mass spectrometry (IDMS): an iterative method, Fresenius J. Anal. Chem. 350 (1994) 657–658. [19] Y. Zhu, K. Chiba, Internal standard method coupled with a gravimetric standard addition method for elemental measurement by ICP-MS, J. Anat. At. Spectrom. 27 (2012) 1000–1006. [20] P. Abbyad, J. Tromp, J. Lam, E. Salin, Optimisation of the technique of standard additions for inductively coupled plasma mass spectrometry, J. Anal. At. Spectrom. 16 (2001) 464–469. [21] S.Y. Ng, Z. Aimei, L.P. Sim, Y. Ding, K.L. Yuen, R.Y.C. Shin, T.K. Lee, High accuracy measurements of trace elements in drinking water by exact matching isotope dilution mass spectrometry (IDMS), Int. J. Mass Spectrom. 321–322 (2012) 19–24. [22] S.Y. Ng, F. Dewi, J. Wang, L.P. Sim, R.Y.C. Shin, T.K. Lee, Development of a cosmetic cream certified reference material: certification of lead: mercury and arsenic mass fractions in cosmetic cream, Int. J. Mass Spectrom. 389 (2015) 59–65. [23] G. Chew, L.P. Sim, S.Y. Ng, Y. Ding, R.Y.C. Shin, T.K. Lee, Development of a mushroom powder certified reference material for calcium, arsenic: cadmium and lead measurements, Food Chem. 190 (2016) 293–299. [24] T. Narukawa, K. Chiba, Heat-assisted aqueous extraction of rice flour for arsenic speciation analysis, J. Agric. Food Chem. 58 (2010) 8183–8188. [25] T. Narukawa, A. Hioki, K. Chiba, Speciation and monitoring test for inorganic arsenic in white rice flour, J. Agric. Food Chem. 60 (2012) 1122–1127.
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001
G Model MASPEC-15703; No. of Pages 7
ARTICLE IN PRESS F. Dewi et al. / International Journal of Mass Spectrometry xxx (2016) xxx–xxx
[26] T. Narukawa, T. Suzuki, K. Inagaki, A. Hioki, Extraction techniques for arsenic species in rice flour and their speciation by HPLC-ICP-MS, Talanta 130 (2014) 213–220. [27] J.-M. Mermet, Limit of quantification in atomic spectrometry: an ambiguous concept? Spectrochim. Acta B 63 (2008) 166–182. [28] F.E. Grubbs, Procedures for detecting outlying observations in samples, Technometrics 11 (1969) 1–21.
7
[29] R.B. Dean, W.J. Dixon, Simplified statistics for small numbers of observations, Anal. Chem. 23 (1951) 636–638. [30] M. Berglund, M.E. Wieser, Isotopic compositions of the elements 2009 (IUPAC Technical Report), Pure Appl. Chem. 83 (2011) 397–410. [31] Joint Committee for Guides in Metrology, JCGM 100:2008 Evaluation of Measurement Data – Guide to the Expression of Uncertainty in Measurement, JCGM, France, 2008.
Please cite this article in press as: F. Dewi, et al., Development of an apple juice certified reference material for cadmium, lead, total arsenic and arsenic species, Int. J. Mass Spectrom. (2016), http://dx.doi.org/10.1016/j.ijms.2016.11.001