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Determination of carbon in digested samples and amino acids by inductively coupled plasma tandem mass spectrometry
Microchemical Journal 122 (2015) 29–32
Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Determination of carbon in digested samples and amino acids by inductively coupled plasma tandem mass spectrometry Clarice D.B. Amaral a,b,⁎, Renata S. Amais c, Lucimar L. Fialho a, Daniela Schiavo d, Ana Rita A. Nogueira b, Joaquim A. Nóbrega a a
Group of Applied Instrumental Analysis, Department of Chemistry, Federal University of São Carlos, P.O. Box 676, São Carlos, SP 13565-905, Brazil Embrapa Southeast Livestock, P.O. Box 339, São Carlos, SP 13560-970, Brazil Center for Nuclear Energy in Agriculture, University of São Paulo, Piracicaba, SP 13416-000, Brazil d Agilent Technologies Brazil, Barueri, SP 06460-040, Brazil b c
a r t i c l e
i n f o
Article history: Received 29 January 2015 Received in revised form 24 March 2015 Accepted 3 April 2015 Available online 10 April 2015 Keywords: Carbon determination ICP-MS/MS Amino acids Mass shift mode Non-metal determination
a b s t r a c t Inductively coupled plasma mass spectrometer with a triple quadrupole (ICP-MS/MS) was employed to determine carbon from different sources such as plant digests, amino acids and peptides. Adequate precision, accuracy and sensitivity were obtained when using either single quadrupole or mass shift mode. Recoveries for carbon determination in plant digests ranged from 96 to 100%. Limits of detection were 0.42 and 0.17 mg L−1 for 12C+ and 12C16O+, respectively, with calibration curves generated by using solutions prepared with oxalic acid. The determination of carbon and sulfur in amino acids and peptides showed that accuracy was better when measuring in O2 reaction mode. Additionally, C and S were simultaneously determined in cystine and glutathione by ICP-MS/ MS mass-shift mode. The determination of carbon simultaneously with trace elements and target elements by ICP-MS makes this concept quite promising. Results demonstrated the potential of ICP-MS/MS for carbon determination. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Carbon determination is important because it is an element present in a wide variety of samples and matrices. Carbon can be bound to trace metals, metalloids, or semimetals and it plays an important role in live organisms [1]. From an analytical point of view, the residual carbon content (RCC) after sample digestion is one of the main parameters to assess the efficiency of decomposition [2]. The efficiency of the sample decomposition in a focused microwave-assisted oven was checked by RCC determination by inductively coupled plasma optical emission spectrometry (ICPOES) with axial viewing [3]. These authors highlighted that for elemental determinations by ICP-MS, knowing the carbon content is important due to several spectral interferences formed with 12C+ and 13 + C [3]. Carbon-based polyatomic ion interferences are so pronounced in ICP-MS that they may hamper the determination of several isotopes, e.g. 40Ar 12C+ overlap on 52Cr+ in biofluid analysis [4]. The use of ICP OES for C, H and O determination in organic compounds such as glucosamine also was pointed out Odenigbo et al. [5]. Detection limits for C, N, H and O were, respectively, 0.2, 50, 1000 and 2000 μmol g−1 of solution and these authors stressed the feasibility of accurately determining C, H and O simultaneously with trace elements [5]. On the other hand, ⁎ Corresponding author. Tel.: +55 16 3351 8058. E-mail address: [email protected] (C.D.B. Amaral).
http://dx.doi.org/10.1016/j.microc.2015.04.007 0026-265X/© 2015 Elsevier B.V. All rights reserved.
carbon in soft drinks was directly determined by ETV-ICPOES using the standard additions method. This procedure could also be employed for determining the carbon content of compounds, such as amino acids, after separation using high performance liquid chromatography, HPLCICPOES [6]. Peters et al. determined carbon in carbohydrates of juice samples by ICP OES following an HPLC separation procedure. The carbon emission detector provided similar detection limits for compounds having similar contents of carbon and they concluded that for analytes having low molar absorptivity in the UV–visible region, the best alternative would be a carbon emission detector [7]. Carrilho et al. determined RCC in forage and protein- and fat-rich materials derived from animal samples after microwave-assisted acid digestion under high pressure in a closed vessel. Determinations were carried out by ICP OES with radial view configuration and correlation between RCC and fat content was found. However, no correlation was observed between RCC and content of protein. The authors inferred that protein-rich samples could be decomposed as efficiently as samples with low protein contents. Based on the easier digestion of proteins compared to fats, they also supposed that the remaining carbon was from fat compounds instead of protein compounds [8]. Interesting applications were proposed for the determination of carbon as a heteroatom-tagged by ICP OES as usually performed for S, P and Se [1]. Morita et al. published the first HPLC-ICP OES study for carbon determination in metalloproteins using size exclusion chromatography [9]. Carbon, P and Co were simultaneously determined in seven
30
C.D.B. Amaral et al. / Microchemical Journal 122 (2015) 29–32
proteins. The carbon source used to prepare analytical calibration solutions was glycylglycine. Peters and Jones [10] published a comprehensive review describing applications of HPLC-ICP OES for C determination in compounds like metalloproteins, amino acids, saccharides, alcohols, caramel colors, and carbohydrates. Despite these antecedents using ICP OES, there are no systematic reports in the literature about quantitative determination of carbon by ICP-MS. Nardi et al. described a simple method for the determination of elements in foods digested with different mixtures of acids and RCCs were determined by measuring the intensities of 12C+ [11]. In other study, the influence of carbon on the signal of some hardto-ionize elements in ICP-MS was investigated and for As, Au, Hg, I, P, Sb, Se, and Te sensitivities were better for carbon-containing solutions compared with those without carbon [12]. On the other hand, no matrix effects were observed for B, Be, Cd, Ir, Os, Pd, Pt, S, and Zn [12]. Addition of carbon in aqueous medium as methanol or ammonium carbonate was made to increase As and Se signals intensities. A solution containing 3% v v−1 methanol caused an increase of 3.5- and 4.5-fold signal intensities for As and Se, respectively, when compared with carbon-free aqueous solutions [13]. The advantage of ICP-MS compared to ICP OES is that generally metals that would be associated to carbon-containing compounds are present at extremely low concentrations, thus C and other elements could be simultaneously determined by ICP-MS due to its superb sensitivity. Fernández et al. demonstrated the highly sensitive simultaneous determination of S and P-peptides without requiring specific standards. The generic standard chosen was bis(4-nitrophenyl)phosphate for P and methionine for S. The species 47P16O+ and 48S16O+ could be detected after the reaction with O2 and these chemically pure compounds would be advantageous according to the authors for use as generic standards in absolute phosphopeptide and S-containing peptide quantification. The detection limits obtained were the lowest ever reported for S- and P-containing species for proteomics analysis, i.e. 0.18 ng S mL−1 and 0.10 ng P mL−1, demonstrating the potential of the approach for proteomics studies [14]. The use of carbon as internal standard in laser ablation-ICP-MS (LA-ICP-MS) analysis is also important. According to Frick and Günther, despite the controversial results found in the literature, carbon as internal standard has been frequently used in carbon-rich sample analysis by LA-ICP-MS and limited attention is given to the difference on ablation behaviors of carbon from synthetic standards and solid samples [15]. Resano et al. evaluated the feasibility of using carbon as internal standard for the multi-element analysis of polymers and they observed losses of precision [16]. In addition to the transport aspects involved in laser ablation analysis, authors attributed this behavior to the high ionization potential (11.3 eV) of carbon which may result in significant signal intensity fluctuations with even minimal variation of plasma conditions. The work here described deals with a systematic study for the determination of carbon by ICP-MS. Firstly, ICP-MS/MS was employed in the carbon determination in different organic compounds and then for RCC determination in plant digests. Later on, it was evaluated the possibility of C and S simultaneous determination in amino acids and peptides.
2. Material and methods
Table 1 Agilent 8800 ICP-MS/MS operating parameters. Instrument parameter
Operating condition
RF applied power (kW) Sampling depth (mm) Number of replicates Stabilization time (s) Auxiliary gas flow rate (L min−1) Makeup gas flow rate (L min−1) Carrier gas flow rate (L min−1) Nebulizer Spray chamber ORS3 cell gas O2 gas flow rate (mL min−1) Selected mass at Q1 Selected mass at Q2
1.55 7 3 10 1.8 0.15 1.05 Concentric nebulizer—glass Scott type—double pass O2 0.30 and 0.75 12 + 13 + 40 C , C , Ar12C+ and 32S+ 12 16 + 13 16 + C O , C O and 32S16O+
2.2. Reagents and analytical solutions Deionized water (resistivity higher than 18.2 MΩ cm) obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to prepare all solutions. Nitric acid (Merck, Darmstadt, Germany) previously purified by a sub-boiling distillation apparatus (Milestone) and 30% m m−1 H2O2 (Labsynth, Diadema, SP, Brazil) were used to digest the samples. Oxalic acid dehydrate (Mallinckrodt Chemicals, St. Louis, MO, USA) and stock monoelement solution containing 1000 mg L−1 of S (Tec-Lab, Hexis, São Paulo, SP, Brazil) were used to prepare C and S reference solutions to build up analytical calibration curves by diluting adequate volumes of inorganic standard solutions in 1% v v− 1 HNO3. Ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), citric acid (Labsynth), glucose (Labsynth), and urea (Reagen, Rio de Janeiro, RJ, Brazil) were used to check the accuracy of the procedure. Carbon and S were also determined in peptides and amino acids: glutathione (Sigma-Aldrich), cystine (99% purity) and leucine (99% purity) obtained from a homeopathic pharmacy located in São Carlos, SP, Brazil. 2.3. Sample preparation Approximately 0.2 g aliquots of standard reference materials (SRM 1515—apple leaves and SRM 1573a—tomato leaves, National Institute of Standards and Technology, Gaithersburg, MD, USA) were accurately weighted directly into the PFA microwave closed vessel and 6 mL HNO3 50% v v−1 and 2 mL H2O2 30% m m−1 were added. The microwave heating program was applied as follows: (1) 20 min to reach 200 °C, (2) 15 min hold at 200 °C. Temperature was controlled by an internal temperature sensor. Vessels were allowed to cool down and final volumes were made up to 10 mL. No solid residues were left in the colorless final sample digests. The procedure was performed in triplicate and a 10-fold dilution was carried out before ICP-MS/MS measurements to ensure dissolved solids content lower than 0.1% m v−1. Before the introduction into ICP-MS/MS, digested samples were degassed using an ultrasonic water bath (Unique USC1400, Indaiatuba, SP, Brazil) for 15 min in order to remove dissolved CO2. Total carbon analyses were carried out in an element analyzer of CHNS-O EA 1108 (Fisons Instruments, Milan, Italy).
2.1. Instrumentation 2.4. Carbon determination from different C sources All measurements were carried out using an inductively coupled plasma tandem mass spectrometer (ICP-MS/MS, Agilent 8800, Tokyo, Japan). The sample introduction system is composed of a Peltier cooled double-pass Scott-type spray chamber and a glass concentric nebulizer. Table 1 shows the ICP-MS/MS operating parameters for both single quadrupole and MS/MS mass-shift modes. An Ethos model 1600 microwave oven was used for sample digestion (Milestone, Sorisole, Italy).
Reference solutions containing 0.05% m v−1 C from ascorbic acid, citric acid, glucose, and urea were prepared and C concentrations were determined by single quadrupole and mass-shift MS/MS modes. External calibration method was performed by using oxalic acid reference solutions ranging from 0.01 to 0.1% m v− 1. Addition and recovery experiments were carried out by adding 0.01 and 0.05% of C to acid digested samples. Carbon and S concentrations were also determined
C.D.B. Amaral et al. / Microchemical Journal 122 (2015) 29–32
in cystine, glutathione, and leucine amino acid solutions. For amino acids, solutions were prepared in order to reach a carbon concentration of 100 mg L−1, therefore, according to their molecular formula should be found 26.8 and 91.1 mg L−1 of sulfur in glutathione and cystine solutions, respectively.
Table 3 Addition and recovery experiments for carbon determination in plant digested samples (mean ± standard deviations, n = 3) in single quadrupole mode. Standard reference material
Added (%)
Single Q—12C+
Rec. (%)
Digested apple leaves (SRM 1515)
0.0100 0.0500 0.0100 0.0500
0.0100 ± 0.0002 0.0498 ± 0.0015 0.0100 ± 0.0002 0.0480 ± 0.002
100 99.6 100 96.0
Digested tomato leaves (SRM 1573a)
3. Results and discussion Some advantages of carbon determination in the same run with other analytes by ICP-MS can be pointed out, however, there is no report in the literature for this analytical strategy. Thus, some possible applications will be discussed here. The first study was concerned to carbon determination from different sources in synthetic solutions and in plant digested samples. Analytical calibration curve for C (linear range from 0.001 to 0.1% m v−1 C) was built up using oxalic acid as the C source. Calibration plots were linear (r2 N 0.99) for all isotopes, species and conditions evaluated. In order to check the performance of the procedure, carbon was determined in solutions containing 0.05 % m v−1 C as urea, glucose, citric acid or ascorbic acid. The determination of C in different carbon compounds (Table 2) showed good accuracy in single quadrupole mode by monitoring both 12C and 13C isotopes. Recoveries ranged from 93.1 to 108% for all solutions analyzed. Sodium carbonate solution was also evaluated to obtain calibration curves but did not present good linearity probably because of formation of CO2 in acid medium. Using ICP OES Gouveia et al. showed that the carbon source (urea, l-cysteine or glucose) and the sample medium (H2SO4, HNO3 or HNO3/H2O2) did not significantly affect the emission intensities when RCC was determined in digested samples, but Y was added as internal standard to correct for transport interferences in H2SO4 medium [17]. Addition and recovery experiments for C in plant sample digests were carried out in two levels (Table 3) in order to check the accuracy of the procedure and the possibility of carbon determination in acid digested samples. As previously mentioned, the determination of carbon by ICP OES is well established [2,3,8], but in case of elemental analysis by ICP-MS, the RCC determination could be simultaneously performed increasing the analytical throughput, and analysts would have the possibility of checking the efficiency of digestions and infer about interferences caused by C-based polyatomic ions. Original carbon contents varied from 37.4% in apple leaves to 38.0% in tomato leaves standard reference materials and the RCCs after microwave-assisted acid digestion were 5.35 and 7.89%, respectively. As can be seen in Table 3, recoveries were quantitative in single quadrupole mode. The great advantage of RCC determination by ICP-MS instead of ICP OES is the determination of C and trace analytes in the same run of analysis. In case of trace element analysis (μg L−1 or ng L−1) ICP OES technique has not enough sensitivity for some elements. Despite not being already demonstrated, the quantitative determination of carbon by ICP-MS is feasible as shown here. Limits of detection (LOD) were calculated as 3 times the standard deviation for 10 consecutive measurements of blank solutions, divided by the slope of the calibration curve. The LOD values found were 0.42,
Table 2 Determination of carbon in different organic C sources (mean ± standard deviations, n = 3) by ICP-MS/MS operated in single quadrupole mode. Single quadrupole mode = 13C+
Carbon source (0.05% m v−1 C)
Single quadrupole mode = 12C+ Found (%)
Rec. (%)
Found (%)
Rec. (%)
Urea Glucose Citric acid Ascorbic acid
0.050 ± 0.001 0.049 ± 0.001 0.051 ± 0.001 0.054 ± 0.001
100 98.7 102 108
0.047 ± 0.001 0.048 ± 0.001 0.047 ± 0.001 0.052 ± 0.001
94.1 96.8 93.1 104
31
0.23, 0.17 and 0.53 mg L−1 for 12C+, 13C+, 12C16O+, and 13C16O+, respectively. The LODs found were considerably better than any previously reported in the literature when C determination was performed by ICP OES as well as background equivalent concentrations (BEC) of 6.1, 6.5, 7.4 and 7.4 mg L− 1, respectively. Gouveia et al. obtained LODs of 33 and 34 mg L−1 C by ICP OES with radial and axial configurations [17], respectively. Carrilho et al. reported that the LOD for C obtained by ICP OES using the 193.027 nm emission line was 25 mg L−1 [8]. Young and Jones calculated 2 mg L− 1 as LOD by ETV-ICP OES [6] and Peters et al. 1.5 mg L−1 carbon for the separation and detection of amino acids by HPLC-ICP OES [18]. Fernandez et al. [14] stressed that higher detection limits usually obtained for S in ICP-MS makes difficult to determine S as a target elements in S-containing peptides. Sulfur and P are used as a target element in speciation analysis for determinations in proteins, amino acids and peptide determinations [19,20]. Bettmer et al. highlighted measurements using a “hard” ion source, such as an ICP-MS, in order to provide elemental and/or isotopic information. According to these authors, the use of an energetic source as the ICP for ionization of proteins and peptides allows for a sensitive and robust determination of an intended element, excepting C, H, O or N, in proteins [21]. In Table 4 are shown C and S determinations in amino acids and peptides by single quadrupole mode and mass-shift mode. This is the second application for carbon determination by ICP-MS addressed in this study. It can be noticed that better accuracy was reached in O2 reaction mode using a 0.75 mL min−1 flow rate. When introducing 0.30 mL min−1 of O2 gas flow rate into the octopole reaction system (ORS3) the 12C16O+ recoveries obtained for cystine, glutathione and leucine were 81.7%, 88.4% and 90.0%, respectively. These values are lower than those obtained using 0.75 mL min−1 O2 (Table 4). In addition, higher O2 gas flow rate into the ORS3 cell can be a favorable medium for the oxide formation and better analytical performance was reported for elements such as S and Si when using higher O2 gas flow rate into the ORS3 in determinations by ICP-MS/MS [22]. Sulfur determinations were also performed, since it is frequently used as a target isotope in metallomic analysis. Single quadrupole mode is not adequate for 32S+ determination at low concentrations due to oxygen based-polyatomic ion interferences. In addition, cystine presents higher sulfur concentration than glutathione, which could explain the low recovery of 32S+ for glutathione. The goal of this experiment was to demonstrate the feasibility of determination of C and S in the same run by ICP-MS/MS increasing the analytical capability for others applications in different fields, such as, studies in proteomics and metabolites determination. Adequate precision and accuracy were observed on 12C+ determination, as well as for its oxide ion, 12C16O+. Carbon and S were determined in the same run, and in such cases oxygen introduction should be used, once in ICP-MS based on quadrupole mass analyzers sulfur isotopes determination is complicated without some strategies to minimize spectral interferences, as can be observed, especially for glutathione. It may be inferred that for the type of matrix evaluated, the determination of carbon could be performed in single quadrupole mode. However, depending on the matrix studied and the concomitant elements, the shift mass mode may be required. The approach for proteomic studies was demonstrated by Fernández et al. using ICP-MS/MS for analysis of S- and P-peptides previously separated using on-line chromatography [14].
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Table 4 Carbon and sulfur determinations in amino acids and peptides (mg L−1, mean ± standard deviations, n = 3) by ICP-MS/MS operated in single quadrupole and mass-shift mode using 0.75 mL min−1 O2 into the octopole reaction system (ORS3). Sample
Single quadrupole mode
Mass shift mode
12 +
32 +
C
Cystine Glutathione Leucine
12 +
C → 12C16O+
S
a
a
32 +
S → 32S16O+
a
Found
Rec. (%)
Found
Rec. (%)
Found
Rec. (%)
Found
Rec.a (%)
91.5 ± 0.6 94.8 ± 0.4 104.4 ± 0.2
91.5 94.8 104
98.4 ± 0.5 20.8 ± 0.2 NP
108 77.7 NP
102.0 ± 0.5 103.3 ± 2.5 114.6 ± 1.4
102 103 115
101.3 ± 1.5 25.0 ± 0.2 NP
111 93.2 NP
NP: not present in the compound. a Recovery is calculated based on concentrations of each prepared solution.
4. Conclusions To the best of our knowledge, carbon monitoring by ICP-MS for its quantification purposes has not been systematically reported. The ICPMS/MS employed in the present work showed to be a suitable and promising tool for determination of C in different compounds and also for RCC determination. Furthermore, C and S were successfully determined in amino acids and peptides, inferring the applicability of C as a target element in speciation analysis after HPLC separation, as it is usually done for S and P in proteins, amino acids and peptide determinations. It was demonstrated that C determination can be done by using either single quadrupole mode or mass shift mode depending on the target elements and specific application. Acknowledgments The authors are thankful to grant 2013/26857-5, São Paulo Research Foundation (FAPESP), to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grants 303107/2013-8, 443771/20146 and 304557/2010-2 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), grant 15/2014 for financial support. We also would like to thank the support from Agilent Technologies. References [1] D. Pröfrock, A. Prange, Inductively coupled plasma-mass spectrometry (ICP-MS) for quantitative analysis in environmental and life sciences: a review of challenges, solutions, and trends, Appl. Spectrosc. 66 (2012) 843–868. [2] A. Krushevska, R.M. Barnes, C.J. Amarasiriwaradena, H. Foner, L. Martines, Determination of the residual carbon content by inductively coupled plasma atomic emission spectrometry after decomposition of biological samples, J. Anal. At. Spectrom. 7 (1992) 845–850. [3] L.M. Costa, F.V. Silva, S.T. Gouveia, A.R.A. Nogueira, J.A. Nóbrega, Focused microwave-assisted acid digestion of oils: an evaluation of the residual carbon content, Spectrochim. Acta B 56 (2001) 1981–1985. [4] A. Krushevska, S. Waheed, J.A. Nóbrega, D. Amarisiriwardena, R.M. Barnes, Reducing polyatomic interferences in the ICP-MS determination of chromium and vanadium in biofluids and tissues, Appl. Spectrosc. 52 (1998) 205–211. [5] C. Odenigbo, Y. Makonnem, A. Asfaw, T. Anastassiades, D. Beauchemin, Towards the use of ICP-OES for the elemental analysis of organic compounds such as glucosamine, J. Anal. At. Spectrom. 29 (2014) 454–457. [6] C.G. Young, B.T. Jones, Determination of the total carbon in soft drinks by tungsten coil electrothermal vaporization inductively coupled plasma spectrometry, Microchem. J. 98 (2011) 323–327.
[7] H.L. Peters, K.E. Levine, B.T. Jones, An inductively coupled plasma carbon emission detector for aqueous carbohydrate separations by liquid chromatography, Anal. Chem. 73 (2001) 453–457. [8] E.N.V.M. Carrilho, A.R.A. Nogueira, J.A. Nóbrega, G.B. de Souza, G.M. Cruz, An attempt to correlate fat and protein content of biological samples with residual carbon after microwave-assisted digestion, Fresenius J. Anal. Chem. 371 (2001) 536–540. [9] M. Morita, T. Uehiro, K. Fuwa, Speciation and elemental analysis of mixtures by high performance liquid chromatography with inductively coupled argon plasma emission spectrometric detection, Anal. Chem. 52 (1980) 349–351. [10] H.L. Peters, B.T. Jones, Determination of non-metals by high performance liquid chromatography with inductively coupled plasma detection, Appl. Spectrosc. Rev. 38 (2003) 71–99. [11] E.P. Nardi, F.S. Evangelista, L. Tormen, T.D. SaintPierre, A.J. Curtius, S.S. de Souza, F. Barbosa Jr., The use of inductively coupled plasma mass spectrometry (ICP-MS) for the determination of toxic and essential elements in different types of food samples, Food Chem. 112 (2009) 727–732. [12] G. Grindlay, J. Mora, M. de Loos-Vollebregt, F. Vanhaecke, A systematic study on the influence of carbon on the behavior of hard-to-ionize elements in inductively coupled plasma-mass spectrometry, Spectrochim. Acta B 86 (2013) 42–49. [13] E.H. Larsen, S. Stürup, Carbon-enhanced inductively coupled plasma mass spectrometric detection of arsenic and selenium and its application to arsenic speciation, J. Anal. At. Spectrom. 9 (1994) 1099–1105. [14] S.D. Fernández, N. Sugishama, J.R. Encinar, A. Sanz-Medel, Triple quad ICPMS (ICPQQQ) as a new tool for absolute quantitative proteomics and phosphoproteomics, Anal. Chem. 84 (2012) 5851–5857. [15] D.A. Frick, D. Günther, Fundamental studies on the ablation behavior of carbon in LA-ICP-MS with respect to the suitability as internal standard, J. Anal. At. Spectrom. 27 (2012) 1294–1303. [16] M. Resano, E. García-Ruiz, F. Vanhaecke, Laser ablation-inductively coupled plasmadynamic reaction cell-mass spectrometry for the multi-element analysis of polymers, Spectrochim. Acta B 60 (2005) 1472–1481. [17] S.T. Gouveia, F.V. Silva, L.M. Costa, A.R.A. Nogueira, J.A. Nóbrega, Determination of residual carbon by inductively-coupled plasma optical emission spectrometry with axial and radial view configurations, Anal. Chim. Acta 445 (2001) 269–275. [18] H.L. Peters, X. Hou, B.T. Jones, Multi-analyte calibration curve for high-performance liquid chromatography with an inductively coupled plasma carbon emission detector, Appl. Spectrosc. 57 (2003) 1162–1166. [19] R. Lobinski, D. Schaumlöffel, J. Szpunar, Mass spectrometry in bioinorganic analytical chemistry, Mass Spectrom. Rev. 25 (2006) 255–289. [20] A. Tholey, D. Schaumlöffel, Metal labeling for quantitative protein and proteome analysis using inductively-coupled plasma mass spectrometry, Trends Anal. Chem. 29 (2010) 399–408. [21] J. Bettmer, M.M. Bayón, J.R. Encinar, M.L.F. Sánchez, M.R.F. de la Campa, A. Sanz-Medel, The emerging role of ICP-MS in proteomic analysis, J. Proteomics 72 (2009) 989–1005. [22] R.S. Amais, C.D.B. Amaral, L.L. Fialho, D. Schiavo, J.A. Nóbrega, Determination of P, S and Si in biodiesel, diesel and lubricating oil using ICP-MS/MS, Anal. Methods 6 (2014) 4516–4520.
Contents lists available at ScienceDirect
Microchemical Journal journal homepage: www.elsevier.com/locate/microc
Determination of carbon in digested samples and amino acids by inductively coupled plasma tandem mass spectrometry Clarice D.B. Amaral a,b,⁎, Renata S. Amais c, Lucimar L. Fialho a, Daniela Schiavo d, Ana Rita A. Nogueira b, Joaquim A. Nóbrega a a
Group of Applied Instrumental Analysis, Department of Chemistry, Federal University of São Carlos, P.O. Box 676, São Carlos, SP 13565-905, Brazil Embrapa Southeast Livestock, P.O. Box 339, São Carlos, SP 13560-970, Brazil Center for Nuclear Energy in Agriculture, University of São Paulo, Piracicaba, SP 13416-000, Brazil d Agilent Technologies Brazil, Barueri, SP 06460-040, Brazil b c
a r t i c l e
i n f o
Article history: Received 29 January 2015 Received in revised form 24 March 2015 Accepted 3 April 2015 Available online 10 April 2015 Keywords: Carbon determination ICP-MS/MS Amino acids Mass shift mode Non-metal determination
a b s t r a c t Inductively coupled plasma mass spectrometer with a triple quadrupole (ICP-MS/MS) was employed to determine carbon from different sources such as plant digests, amino acids and peptides. Adequate precision, accuracy and sensitivity were obtained when using either single quadrupole or mass shift mode. Recoveries for carbon determination in plant digests ranged from 96 to 100%. Limits of detection were 0.42 and 0.17 mg L−1 for 12C+ and 12C16O+, respectively, with calibration curves generated by using solutions prepared with oxalic acid. The determination of carbon and sulfur in amino acids and peptides showed that accuracy was better when measuring in O2 reaction mode. Additionally, C and S were simultaneously determined in cystine and glutathione by ICP-MS/ MS mass-shift mode. The determination of carbon simultaneously with trace elements and target elements by ICP-MS makes this concept quite promising. Results demonstrated the potential of ICP-MS/MS for carbon determination. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Carbon determination is important because it is an element present in a wide variety of samples and matrices. Carbon can be bound to trace metals, metalloids, or semimetals and it plays an important role in live organisms [1]. From an analytical point of view, the residual carbon content (RCC) after sample digestion is one of the main parameters to assess the efficiency of decomposition [2]. The efficiency of the sample decomposition in a focused microwave-assisted oven was checked by RCC determination by inductively coupled plasma optical emission spectrometry (ICPOES) with axial viewing [3]. These authors highlighted that for elemental determinations by ICP-MS, knowing the carbon content is important due to several spectral interferences formed with 12C+ and 13 + C [3]. Carbon-based polyatomic ion interferences are so pronounced in ICP-MS that they may hamper the determination of several isotopes, e.g. 40Ar 12C+ overlap on 52Cr+ in biofluid analysis [4]. The use of ICP OES for C, H and O determination in organic compounds such as glucosamine also was pointed out Odenigbo et al. [5]. Detection limits for C, N, H and O were, respectively, 0.2, 50, 1000 and 2000 μmol g−1 of solution and these authors stressed the feasibility of accurately determining C, H and O simultaneously with trace elements [5]. On the other hand, ⁎ Corresponding author. Tel.: +55 16 3351 8058. E-mail address: [email protected] (C.D.B. Amaral).
http://dx.doi.org/10.1016/j.microc.2015.04.007 0026-265X/© 2015 Elsevier B.V. All rights reserved.
carbon in soft drinks was directly determined by ETV-ICPOES using the standard additions method. This procedure could also be employed for determining the carbon content of compounds, such as amino acids, after separation using high performance liquid chromatography, HPLCICPOES [6]. Peters et al. determined carbon in carbohydrates of juice samples by ICP OES following an HPLC separation procedure. The carbon emission detector provided similar detection limits for compounds having similar contents of carbon and they concluded that for analytes having low molar absorptivity in the UV–visible region, the best alternative would be a carbon emission detector [7]. Carrilho et al. determined RCC in forage and protein- and fat-rich materials derived from animal samples after microwave-assisted acid digestion under high pressure in a closed vessel. Determinations were carried out by ICP OES with radial view configuration and correlation between RCC and fat content was found. However, no correlation was observed between RCC and content of protein. The authors inferred that protein-rich samples could be decomposed as efficiently as samples with low protein contents. Based on the easier digestion of proteins compared to fats, they also supposed that the remaining carbon was from fat compounds instead of protein compounds [8]. Interesting applications were proposed for the determination of carbon as a heteroatom-tagged by ICP OES as usually performed for S, P and Se [1]. Morita et al. published the first HPLC-ICP OES study for carbon determination in metalloproteins using size exclusion chromatography [9]. Carbon, P and Co were simultaneously determined in seven
30
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proteins. The carbon source used to prepare analytical calibration solutions was glycylglycine. Peters and Jones [10] published a comprehensive review describing applications of HPLC-ICP OES for C determination in compounds like metalloproteins, amino acids, saccharides, alcohols, caramel colors, and carbohydrates. Despite these antecedents using ICP OES, there are no systematic reports in the literature about quantitative determination of carbon by ICP-MS. Nardi et al. described a simple method for the determination of elements in foods digested with different mixtures of acids and RCCs were determined by measuring the intensities of 12C+ [11]. In other study, the influence of carbon on the signal of some hardto-ionize elements in ICP-MS was investigated and for As, Au, Hg, I, P, Sb, Se, and Te sensitivities were better for carbon-containing solutions compared with those without carbon [12]. On the other hand, no matrix effects were observed for B, Be, Cd, Ir, Os, Pd, Pt, S, and Zn [12]. Addition of carbon in aqueous medium as methanol or ammonium carbonate was made to increase As and Se signals intensities. A solution containing 3% v v−1 methanol caused an increase of 3.5- and 4.5-fold signal intensities for As and Se, respectively, when compared with carbon-free aqueous solutions [13]. The advantage of ICP-MS compared to ICP OES is that generally metals that would be associated to carbon-containing compounds are present at extremely low concentrations, thus C and other elements could be simultaneously determined by ICP-MS due to its superb sensitivity. Fernández et al. demonstrated the highly sensitive simultaneous determination of S and P-peptides without requiring specific standards. The generic standard chosen was bis(4-nitrophenyl)phosphate for P and methionine for S. The species 47P16O+ and 48S16O+ could be detected after the reaction with O2 and these chemically pure compounds would be advantageous according to the authors for use as generic standards in absolute phosphopeptide and S-containing peptide quantification. The detection limits obtained were the lowest ever reported for S- and P-containing species for proteomics analysis, i.e. 0.18 ng S mL−1 and 0.10 ng P mL−1, demonstrating the potential of the approach for proteomics studies [14]. The use of carbon as internal standard in laser ablation-ICP-MS (LA-ICP-MS) analysis is also important. According to Frick and Günther, despite the controversial results found in the literature, carbon as internal standard has been frequently used in carbon-rich sample analysis by LA-ICP-MS and limited attention is given to the difference on ablation behaviors of carbon from synthetic standards and solid samples [15]. Resano et al. evaluated the feasibility of using carbon as internal standard for the multi-element analysis of polymers and they observed losses of precision [16]. In addition to the transport aspects involved in laser ablation analysis, authors attributed this behavior to the high ionization potential (11.3 eV) of carbon which may result in significant signal intensity fluctuations with even minimal variation of plasma conditions. The work here described deals with a systematic study for the determination of carbon by ICP-MS. Firstly, ICP-MS/MS was employed in the carbon determination in different organic compounds and then for RCC determination in plant digests. Later on, it was evaluated the possibility of C and S simultaneous determination in amino acids and peptides.
2. Material and methods
Table 1 Agilent 8800 ICP-MS/MS operating parameters. Instrument parameter
Operating condition
RF applied power (kW) Sampling depth (mm) Number of replicates Stabilization time (s) Auxiliary gas flow rate (L min−1) Makeup gas flow rate (L min−1) Carrier gas flow rate (L min−1) Nebulizer Spray chamber ORS3 cell gas O2 gas flow rate (mL min−1) Selected mass at Q1 Selected mass at Q2
1.55 7 3 10 1.8 0.15 1.05 Concentric nebulizer—glass Scott type—double pass O2 0.30 and 0.75 12 + 13 + 40 C , C , Ar12C+ and 32S+ 12 16 + 13 16 + C O , C O and 32S16O+
2.2. Reagents and analytical solutions Deionized water (resistivity higher than 18.2 MΩ cm) obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used to prepare all solutions. Nitric acid (Merck, Darmstadt, Germany) previously purified by a sub-boiling distillation apparatus (Milestone) and 30% m m−1 H2O2 (Labsynth, Diadema, SP, Brazil) were used to digest the samples. Oxalic acid dehydrate (Mallinckrodt Chemicals, St. Louis, MO, USA) and stock monoelement solution containing 1000 mg L−1 of S (Tec-Lab, Hexis, São Paulo, SP, Brazil) were used to prepare C and S reference solutions to build up analytical calibration curves by diluting adequate volumes of inorganic standard solutions in 1% v v− 1 HNO3. Ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA), citric acid (Labsynth), glucose (Labsynth), and urea (Reagen, Rio de Janeiro, RJ, Brazil) were used to check the accuracy of the procedure. Carbon and S were also determined in peptides and amino acids: glutathione (Sigma-Aldrich), cystine (99% purity) and leucine (99% purity) obtained from a homeopathic pharmacy located in São Carlos, SP, Brazil. 2.3. Sample preparation Approximately 0.2 g aliquots of standard reference materials (SRM 1515—apple leaves and SRM 1573a—tomato leaves, National Institute of Standards and Technology, Gaithersburg, MD, USA) were accurately weighted directly into the PFA microwave closed vessel and 6 mL HNO3 50% v v−1 and 2 mL H2O2 30% m m−1 were added. The microwave heating program was applied as follows: (1) 20 min to reach 200 °C, (2) 15 min hold at 200 °C. Temperature was controlled by an internal temperature sensor. Vessels were allowed to cool down and final volumes were made up to 10 mL. No solid residues were left in the colorless final sample digests. The procedure was performed in triplicate and a 10-fold dilution was carried out before ICP-MS/MS measurements to ensure dissolved solids content lower than 0.1% m v−1. Before the introduction into ICP-MS/MS, digested samples were degassed using an ultrasonic water bath (Unique USC1400, Indaiatuba, SP, Brazil) for 15 min in order to remove dissolved CO2. Total carbon analyses were carried out in an element analyzer of CHNS-O EA 1108 (Fisons Instruments, Milan, Italy).
2.1. Instrumentation 2.4. Carbon determination from different C sources All measurements were carried out using an inductively coupled plasma tandem mass spectrometer (ICP-MS/MS, Agilent 8800, Tokyo, Japan). The sample introduction system is composed of a Peltier cooled double-pass Scott-type spray chamber and a glass concentric nebulizer. Table 1 shows the ICP-MS/MS operating parameters for both single quadrupole and MS/MS mass-shift modes. An Ethos model 1600 microwave oven was used for sample digestion (Milestone, Sorisole, Italy).
Reference solutions containing 0.05% m v−1 C from ascorbic acid, citric acid, glucose, and urea were prepared and C concentrations were determined by single quadrupole and mass-shift MS/MS modes. External calibration method was performed by using oxalic acid reference solutions ranging from 0.01 to 0.1% m v− 1. Addition and recovery experiments were carried out by adding 0.01 and 0.05% of C to acid digested samples. Carbon and S concentrations were also determined
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in cystine, glutathione, and leucine amino acid solutions. For amino acids, solutions were prepared in order to reach a carbon concentration of 100 mg L−1, therefore, according to their molecular formula should be found 26.8 and 91.1 mg L−1 of sulfur in glutathione and cystine solutions, respectively.
Table 3 Addition and recovery experiments for carbon determination in plant digested samples (mean ± standard deviations, n = 3) in single quadrupole mode. Standard reference material
Added (%)
Single Q—12C+
Rec. (%)
Digested apple leaves (SRM 1515)
0.0100 0.0500 0.0100 0.0500
0.0100 ± 0.0002 0.0498 ± 0.0015 0.0100 ± 0.0002 0.0480 ± 0.002
100 99.6 100 96.0
Digested tomato leaves (SRM 1573a)
3. Results and discussion Some advantages of carbon determination in the same run with other analytes by ICP-MS can be pointed out, however, there is no report in the literature for this analytical strategy. Thus, some possible applications will be discussed here. The first study was concerned to carbon determination from different sources in synthetic solutions and in plant digested samples. Analytical calibration curve for C (linear range from 0.001 to 0.1% m v−1 C) was built up using oxalic acid as the C source. Calibration plots were linear (r2 N 0.99) for all isotopes, species and conditions evaluated. In order to check the performance of the procedure, carbon was determined in solutions containing 0.05 % m v−1 C as urea, glucose, citric acid or ascorbic acid. The determination of C in different carbon compounds (Table 2) showed good accuracy in single quadrupole mode by monitoring both 12C and 13C isotopes. Recoveries ranged from 93.1 to 108% for all solutions analyzed. Sodium carbonate solution was also evaluated to obtain calibration curves but did not present good linearity probably because of formation of CO2 in acid medium. Using ICP OES Gouveia et al. showed that the carbon source (urea, l-cysteine or glucose) and the sample medium (H2SO4, HNO3 or HNO3/H2O2) did not significantly affect the emission intensities when RCC was determined in digested samples, but Y was added as internal standard to correct for transport interferences in H2SO4 medium [17]. Addition and recovery experiments for C in plant sample digests were carried out in two levels (Table 3) in order to check the accuracy of the procedure and the possibility of carbon determination in acid digested samples. As previously mentioned, the determination of carbon by ICP OES is well established [2,3,8], but in case of elemental analysis by ICP-MS, the RCC determination could be simultaneously performed increasing the analytical throughput, and analysts would have the possibility of checking the efficiency of digestions and infer about interferences caused by C-based polyatomic ions. Original carbon contents varied from 37.4% in apple leaves to 38.0% in tomato leaves standard reference materials and the RCCs after microwave-assisted acid digestion were 5.35 and 7.89%, respectively. As can be seen in Table 3, recoveries were quantitative in single quadrupole mode. The great advantage of RCC determination by ICP-MS instead of ICP OES is the determination of C and trace analytes in the same run of analysis. In case of trace element analysis (μg L−1 or ng L−1) ICP OES technique has not enough sensitivity for some elements. Despite not being already demonstrated, the quantitative determination of carbon by ICP-MS is feasible as shown here. Limits of detection (LOD) were calculated as 3 times the standard deviation for 10 consecutive measurements of blank solutions, divided by the slope of the calibration curve. The LOD values found were 0.42,
Table 2 Determination of carbon in different organic C sources (mean ± standard deviations, n = 3) by ICP-MS/MS operated in single quadrupole mode. Single quadrupole mode = 13C+
Carbon source (0.05% m v−1 C)
Single quadrupole mode = 12C+ Found (%)
Rec. (%)
Found (%)
Rec. (%)
Urea Glucose Citric acid Ascorbic acid
0.050 ± 0.001 0.049 ± 0.001 0.051 ± 0.001 0.054 ± 0.001
100 98.7 102 108
0.047 ± 0.001 0.048 ± 0.001 0.047 ± 0.001 0.052 ± 0.001
94.1 96.8 93.1 104
31
0.23, 0.17 and 0.53 mg L−1 for 12C+, 13C+, 12C16O+, and 13C16O+, respectively. The LODs found were considerably better than any previously reported in the literature when C determination was performed by ICP OES as well as background equivalent concentrations (BEC) of 6.1, 6.5, 7.4 and 7.4 mg L− 1, respectively. Gouveia et al. obtained LODs of 33 and 34 mg L−1 C by ICP OES with radial and axial configurations [17], respectively. Carrilho et al. reported that the LOD for C obtained by ICP OES using the 193.027 nm emission line was 25 mg L−1 [8]. Young and Jones calculated 2 mg L− 1 as LOD by ETV-ICP OES [6] and Peters et al. 1.5 mg L−1 carbon for the separation and detection of amino acids by HPLC-ICP OES [18]. Fernandez et al. [14] stressed that higher detection limits usually obtained for S in ICP-MS makes difficult to determine S as a target elements in S-containing peptides. Sulfur and P are used as a target element in speciation analysis for determinations in proteins, amino acids and peptide determinations [19,20]. Bettmer et al. highlighted measurements using a “hard” ion source, such as an ICP-MS, in order to provide elemental and/or isotopic information. According to these authors, the use of an energetic source as the ICP for ionization of proteins and peptides allows for a sensitive and robust determination of an intended element, excepting C, H, O or N, in proteins [21]. In Table 4 are shown C and S determinations in amino acids and peptides by single quadrupole mode and mass-shift mode. This is the second application for carbon determination by ICP-MS addressed in this study. It can be noticed that better accuracy was reached in O2 reaction mode using a 0.75 mL min−1 flow rate. When introducing 0.30 mL min−1 of O2 gas flow rate into the octopole reaction system (ORS3) the 12C16O+ recoveries obtained for cystine, glutathione and leucine were 81.7%, 88.4% and 90.0%, respectively. These values are lower than those obtained using 0.75 mL min−1 O2 (Table 4). In addition, higher O2 gas flow rate into the ORS3 cell can be a favorable medium for the oxide formation and better analytical performance was reported for elements such as S and Si when using higher O2 gas flow rate into the ORS3 in determinations by ICP-MS/MS [22]. Sulfur determinations were also performed, since it is frequently used as a target isotope in metallomic analysis. Single quadrupole mode is not adequate for 32S+ determination at low concentrations due to oxygen based-polyatomic ion interferences. In addition, cystine presents higher sulfur concentration than glutathione, which could explain the low recovery of 32S+ for glutathione. The goal of this experiment was to demonstrate the feasibility of determination of C and S in the same run by ICP-MS/MS increasing the analytical capability for others applications in different fields, such as, studies in proteomics and metabolites determination. Adequate precision and accuracy were observed on 12C+ determination, as well as for its oxide ion, 12C16O+. Carbon and S were determined in the same run, and in such cases oxygen introduction should be used, once in ICP-MS based on quadrupole mass analyzers sulfur isotopes determination is complicated without some strategies to minimize spectral interferences, as can be observed, especially for glutathione. It may be inferred that for the type of matrix evaluated, the determination of carbon could be performed in single quadrupole mode. However, depending on the matrix studied and the concomitant elements, the shift mass mode may be required. The approach for proteomic studies was demonstrated by Fernández et al. using ICP-MS/MS for analysis of S- and P-peptides previously separated using on-line chromatography [14].
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Table 4 Carbon and sulfur determinations in amino acids and peptides (mg L−1, mean ± standard deviations, n = 3) by ICP-MS/MS operated in single quadrupole and mass-shift mode using 0.75 mL min−1 O2 into the octopole reaction system (ORS3). Sample
Single quadrupole mode
Mass shift mode
12 +
32 +
C
Cystine Glutathione Leucine
12 +
C → 12C16O+
S
a
a
32 +
S → 32S16O+
a
Found
Rec. (%)
Found
Rec. (%)
Found
Rec. (%)
Found
Rec.a (%)
91.5 ± 0.6 94.8 ± 0.4 104.4 ± 0.2
91.5 94.8 104
98.4 ± 0.5 20.8 ± 0.2 NP
108 77.7 NP
102.0 ± 0.5 103.3 ± 2.5 114.6 ± 1.4
102 103 115
101.3 ± 1.5 25.0 ± 0.2 NP
111 93.2 NP
NP: not present in the compound. a Recovery is calculated based on concentrations of each prepared solution.
4. Conclusions To the best of our knowledge, carbon monitoring by ICP-MS for its quantification purposes has not been systematically reported. The ICPMS/MS employed in the present work showed to be a suitable and promising tool for determination of C in different compounds and also for RCC determination. Furthermore, C and S were successfully determined in amino acids and peptides, inferring the applicability of C as a target element in speciation analysis after HPLC separation, as it is usually done for S and P in proteins, amino acids and peptide determinations. It was demonstrated that C determination can be done by using either single quadrupole mode or mass shift mode depending on the target elements and specific application. Acknowledgments The authors are thankful to grant 2013/26857-5, São Paulo Research Foundation (FAPESP), to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grants 303107/2013-8, 443771/20146 and 304557/2010-2 and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), grant 15/2014 for financial support. We also would like to thank the support from Agilent Technologies. References [1] D. Pröfrock, A. Prange, Inductively coupled plasma-mass spectrometry (ICP-MS) for quantitative analysis in environmental and life sciences: a review of challenges, solutions, and trends, Appl. Spectrosc. 66 (2012) 843–868. [2] A. Krushevska, R.M. Barnes, C.J. Amarasiriwaradena, H. Foner, L. Martines, Determination of the residual carbon content by inductively coupled plasma atomic emission spectrometry after decomposition of biological samples, J. Anal. At. Spectrom. 7 (1992) 845–850. [3] L.M. Costa, F.V. Silva, S.T. Gouveia, A.R.A. Nogueira, J.A. Nóbrega, Focused microwave-assisted acid digestion of oils: an evaluation of the residual carbon content, Spectrochim. Acta B 56 (2001) 1981–1985. [4] A. Krushevska, S. Waheed, J.A. Nóbrega, D. Amarisiriwardena, R.M. Barnes, Reducing polyatomic interferences in the ICP-MS determination of chromium and vanadium in biofluids and tissues, Appl. Spectrosc. 52 (1998) 205–211. [5] C. Odenigbo, Y. Makonnem, A. Asfaw, T. Anastassiades, D. Beauchemin, Towards the use of ICP-OES for the elemental analysis of organic compounds such as glucosamine, J. Anal. At. Spectrom. 29 (2014) 454–457. [6] C.G. Young, B.T. Jones, Determination of the total carbon in soft drinks by tungsten coil electrothermal vaporization inductively coupled plasma spectrometry, Microchem. J. 98 (2011) 323–327.
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