- Email: [email protected]
Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry
Accepted Manuscript Analytical Methods Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry Laís N. Pires, Geovani C. Brandão, Leonardo S.G. Teixeira PII: DOI: Reference:
S0308-8146(17)30019-5 http://dx.doi.org/10.1016/j.foodchem.2017.01.019 FOCH 20410
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
26 September 2016 3 January 2017 4 January 2017
Please cite this article as: Pires, L.N., Brandão, G.C., Teixeira, L.S.G., Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.01.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry
Laís N. Pires a, Geovani C. Brandãoa,b, Leonardo S. G. Teixeiraa,b*
a
Universidade Federal da Bahia, Instituto de Química, Departamento de
Química Analítica, Campus Universitário de Ondina, 40170-115, Salvador, Bahia, Brasil. b
INCT de Energia e Ambiente - Universidade Federal da Bahia, Instituto de
Química, Campus Universitário de Ondina, 40170-115, Salvador, Bahia, Brasil.
_________________________________________________________________________ *
Corresponding author: e-mail: [email protected]; Tel + 55 71 3283-6830; Fax +55 71 3235-5166
1
Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry
Abstract This paper presents a method for determining phospholipids in soybean lecithin samples by phosphorus determination using high-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS GF MAS) via molecular absorption of phosphorus monoxide. Samples were diluted in methyl isobutyl ketone. The best conditions were found to be 213.561 nm with a pyrolysis temperature of 1300°C, a volatilization temperature of 2300°C and Mg as a chemical modifier. To increase the analytical sensitivity, measurement of the absorbance signal was obtained by summing molecular transition lines for PO surrounding 213 nm: 213.561, 213.526, 213.617 and 213.637 nm. The limit of detection was 2.35 mg g-1 and the precision, evaluated as relative standard deviation (RSD), was 2.47% (n=10) for a sample containing 2.2% (w/v) phosphorus. The developed method was applied for the analysis of commercial samples of soybean lecithin. The determined concentrations of phospholipids in the samples varied between 38.1 and 45% (w/v).
Keywords:
phospholipids;
soybean
lecithin;
phosphorus;
phosphorus
monoxide; HR-CS GF MAS.
2
1. Introduction Lecithin is a mixture found in animal tissues and in vegetable oils, being composed of choline, glycerol, fatty acids, glycolipids, triacylglycerols and, mainly, phospholipids such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, with smaller quantities of phosphatidic acid and phosphatidylserine (Cooper & Ainscough, 2016). Among all vegetable oils, soybean oil contains the greatest amount of lecithin, which is separated from the crude oil by the degumming process. Crude lecithin is a viscous liquid with a color ranging from brown to pale yellow. It contains about 60–70% phospholipids, 30–40% of neutral oil and about 0.5–2% moisture (Rydhag & Wilton, 1981). The phospholipids present in lecithin are essential components of cell membranes and, therefore, essential for the growth, maturation and proper function of cells (Liu, Waters, Rose, Bao & King, 2013). Phosphatidylcholine is present in the greatest quantity in lecithin and makes a direct contribution to the formation of bilipid layers in cell membranes. It is also a precursor of choline synthesis within the body, a nutrient that is essential for the transport of fats and a precursor of acetylcholine — itself an important neurotransmitter necessary for memory and other brain functions (Jangle, Magar & Thorat, 2013). Phospholipids have gained special attention because of some intrinsic health benefits. Studies have suggested the therapeutic use of phospholipids such as lecithin for the improvement of memory, liver protection, cholesterol reduction and treating neurological disorders (Szydłowska-Czerniak, 2007). Furthermore, due to their amphiphilic nature, availability and excellent functionality, phospholipids have wide application as an emulsifier in the food, 3
pharmaceutical and cosmetic industries (Jangle, Magar & Thorat, 2013; Mertins, Sebben, Schneider, Pohlmann & Silveira, 2008). Thus, there is a necessity for analytical methods to monitor the quality of commercial soybean lecithin, in particular related to their phospholipid content. Due to its complex composition, most reports on the analysis of lecithin and fractionation of its phospholipids involve expensive and laborious procedures (Fernandes, Alberici, Pereira, Cabral, Eberlin & Barrera-Arellano, 2012). Various analytical techniques have been used for the determination of phospholipids in soybean lecithin, for example, easy ambient sonic-spray ionization mass spectrometry (EASI-MS) spectroscopy (Fernandes, Alberici, Pereira, Cabral, Eberlin & Barrera-Arellano, 2012), Fourier transform near infrared spectroscopy (FT-NIR) (Li, Goulden, Cocciardi, Hughes, 2009), thinlayer chromatography (TLC) (Helmerich & Koehler, 2003), high-performance liquid chromatography (HPLC) (Helmerich &
Koehler, 2003), (31)P nuclear
magnetic resonance spectroscopy ((31)P NMR) (Helmerich & Koehler, 2003), UV-vis spectroscopy (John, Park, Lee, Suh & Kim, 2015) and colorimetry (Totani, Pretorius & Plessis, 1982). Although phosphorus determination in complex matrices using atomic spectrometric techniques is still challenging, high-resolution continuum source molecular absorption spectrometry (HR-CS MAS) has shown promise for overcoming these difficulties. Molecular absorption spectrometry (MAS) is a technique developed for monitoring the absorption of diatomic molecules such as NO, CS and PO (Welz, Lepri, Araujo, Ferreira, Huang, Okruss & Becker-Ross, 2009). The technique has been employed in the determination of phosphorus via the PO molecule in wheat flour samples (Heitmann, Becker-Ross, Florek, Huang & Okruss, 2006)
4
and biological materials (Resano, Briceño & Belarra, 2009). However, it should be noted that the determination of phospholipids in soybean lecithin samples via the PO molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS GF MAS) has not yet been recorded in the literature. In this study, an analytical method is proposed for the determination of phospholipids in soybean lecithin samples by HR-CS GF MAS via the molecular absorption of phosphorus monoxide. Different organic solvents were tested as diluents to allow the introduction of samples into the spectrometer. Moreover, the temperature conditions for phosphorus determination by PO absorption were studied for different transition lines in the presence and absence of chemical modifiers.
2. Experimental 2.1. Instrumentation All measurements of molecular absorption of PO were performed employing a high-resolution continuum source electrothermal atomic absorption spectrometer model ContrAA 700 (Analytik Jena AG, Jena, Germany). The spectrometer was equipped with a xenon short-arc lamp as a continuum radiation source, a double-echelle monochromator and a charge-coupled device (CCD) array detector. Pyrolytically coated graphite tubes and platforms, provided by the same manufacturer, were transversely heated and used for all determinations. Argon with a purity of 99.999% (White Martins, São Paulo, Brazil) was used as the purge gas at a flow rate of 2.0 L min-1. The integrated
5
peak area using three pixels was employed as the analytical signal. The temperature program used for the determinations is presented in Table 1. As a comparative procedure, the soybean lecithin samples were subjected to acid digestion employing a heating block model TE-040/25 (Tecnal, São Paulo, Brazil). After digestion, an inductively coupled plasma optical emission spectrometry (ICP OES) instrument, model 720 series (Agilent Technologies, Santa Clara, USA) was used to determine the phosphorus content at 213.618 nm. The equipment was operated under the manufacturer’s recommended conditions for power (1.10 kW), plasma gas flow (15 L min-1), auxiliary gas flow (1.5 L min-1) and nebulizer gas flow (0.75 L min-1), using a Sturman-Master chamber and a V-Groove nebulizer.
2.2. Reagents, solutions and samples All chemicals used in the experiments were of analytical grade. The aqueous solutions were prepared using ultrapure water (resistivity > 18 MΩ cm) obtained from a Milli-Q® purification system (Millipore, Bedford, USA). All flasks and glassware were soaked in 10% (v/v) HNO3 for at least 12 h and rinsed with water before use. A 0.1% (w/v) Ca solution and a 0.1% (w/v) Mg solution, used as chemical modifiers, were prepared by dissolving CaCl2 (Merck, Darmstadt, Germany) and Mg(NO3)2 (Merck), respectively, in propan-1-ol (Vetec, Rio de Janeiro, Brazil). A 1000 mg L-1 phosphorus stock solution was prepared by dissolving anhydrous monobasic ammonium phosphate (Merck) in water. Methyl isobutyl ketone (Merck), pentane (Merck), hexane (Merck), acetone (Vetec), acetic acid (Vetec), methylethylketone (Merck), propan-1-ol
6
(Vetec) and methanol (Vetec) were tested as solvents for the dilution of the soybean lecithin samples. The proposed procedure was applied to the analysis of six soybean lecithin samples purchased in markets in Salvador City, Bahia, Brazil.
2.3. Sample preparation Approximately 0.1000 g of each soybean lecithin sample was weighed directly into a polyethylene tube and diluted in 10 mL of MIBK for use in HR-CS GF MAS. All samples were prepared in triplicate. The results obtained with the proposed method were compared with those resulting from samples treated by acid digestion and determination by ICP OES. For ICP OES, approximately 0.2000 g of each sample was weighed directly into a digester. Then, 3.0 mL of concentrated HNO3 and 1.0 mL of concentrated H2SO4 were added. The system temperature was varied in steps: in the first 15 min, the temperature was maintained at 150 °C and then maintained at 180 °C for another 15 min. Afterwards, 2.0 mL of H2O2 30% (v/v) was added and the temperature was raised to 220 °C. The total time of the procedure was 2 h. After cooling to room temperature, the digestion was diluted with water for 10 mL in polyethylene bottles. The digestion of all samples was performed in triplicate and analytical blanks were also obtained in the same way. Working standard solutions for phosphorus were prepared by dilution from 1000 mg L-1 stock solutions (Specsol Quimlab, Brazil).
7
3. Results and discussion 3.1. Sample dilution Aspiration of high viscosity samples into the detection system can be a problem when graphite furnace atomic absorption spectrometry (GF AAS) is employed in elemental determination (Almeida, Brandão, Santos & Teixeira, 2016). Dilution in organic solvents is a method of sample preparation that allows adjustment of the viscosity of the sample, enabling simple and fast analysis of oily matrices by atomic spectrometric techniques. Among the most frequently used solvents for oily sample dilution are MIBK, pentane, hexane, acetone, acetic acid, methylethylketone, propan-1-ol, methanol and toluene (Amorim, Welz, Costa, Lepri, Vale & Ferreira, 2007; Vale, Damin, Klassen, Silva, Welz, Silva, Lepri, Borges & Heitmann, 2004). Because lecithin is composed of phospholipids with different groups that impart different polarity and solubility in organic solvents (John, Park, Lee, Suh & Kim, 2015), several of these solvents were tested for the dilution of lecithin samples. Soybean lecithin samples were partially dissolved in acetone, methyl ethyl ketone and acetic acid. Appropriate solubilization was also not observed when the propane and methanol were used. However, the samples were completely dissolved when MIBK, pentane and hexane were used as diluents. A study of the values and stability of analytical signals was then accomplished by observing the profile of absorption peaks obtained with the use of these three solvents. Stable analytical signals were found for the three solvents after successive measurements. Additionally, the analytical responses were similar when MIBK, pentane or cyclohexane were used; however, narrower peaks and lower background signals were obtained with samples diluted in MIBK. Thus, 8
MIBK was used as the solvent for the dilution of lecithin samples, in agreement with previous work that used this solvent for the dilution of organic matrices for determination by GF AAS (Vale, Damin, Klassen, Silva, Welz, Silva, Lepri, Borges & Heitmann, 2004; Gonzalez, Rodriguez & Gonzalez, 1987; MartínPolvillo, Albi & Guinda, 1994).
3.2. Optimization of the furnace heating program The diatomic PO molecule presents three different electronic transitions and, consequently, has a large number of molecular lines with different sensitivities that can be used for the determination of phosphorus (Welz, 2005). Huang et al. (2006), for example, studied the absorption of this molecule at 246.40, 247.78, 324.62 and 327.04 nm, with attention to possible interference in each line, while Resano et al. (2009) have conducted studies on the absorption of PO exploring the sum of the lines in the spectral region around 213.618 nm. In this work, studies were performed to define the best electrothermal conditions and the best chemical modifier to be used for the lines of molecular absorption of PO at 213.561, 246.400 and 324.620 nm, in order to establish the most appropriate line for the quantification of phospholipids in soybean lecithin samples. Modifiers that do not favor P reduction and atomization were used, because otherwise an spectral interference would appear. A soybean lecithin sample diluted in MIBK was employed, varying pyrolysis and vaporization temperatures between 700-1800 °C and 1700-2700 °C, respectively, in the absence and in the presence of calcium and magnesium as chemical modifiers instead of more traditional modifiers like Pd, which will produce atomic P (Lepri, 9
Dessuy, Vale, Borges, Welz & Heitmann, 2006; Huang, 2006; Resano, 2009; Resano, Belarra, Castillo & Vanhaecke, 2000). The pyrolysis and vaporization curves obtained for each line are presented in Figure 1. The different sensitivities of each absorption line were confirmed with the pyrolysis and vaporization curves, wherein the highest analytical signals at 213.561 nm were obtained in the presence of calcium, while the highest analytical signals at 246.400 and 324.620 nm were obtained in the presence of magnesium. In addition, the results obtained showed that the pyrolysis and vaporization curves were well defined only at 213.561 nm, with analytical signals favored by the presence of the modifiers. These results are consistent with previous work, where difficulties in the optimization of pyrolysis and vaporization temperatures have been reported using the molecular transition lines at 246.400 and 324.620 nm (Dessuy, 2007). A further observation was that vaporization temperatures over 2300 °C can result in a decrease in sensitivity for the determination of PO, which could be the result of factors such as: formation of atomic phosphorus, which begins to be significant at higher temperatures; or the possible formation of other molecular species such as P2 and PO2 (Resano, Briceño & Belarra, 2009). This behavior was observed in the study of temperature of vaporization at 213.561 nm in the presence of the modifiers, where the absorbance signals decreased slightly at temperatures up to 2300 °C. However, these factors did not impair the determination when temperatures up to 2300 °C were employed in the presence of the modifiers. The pyrolysis and vaporization curves at 213.561 nm also showed that the thermal behavior was similar when calcium and magnesium were used as
10
chemical modifiers. Probably the same mechanism of stabilization of the analyte was obtained for both modifiers, which indicates that either of them could be used for the determination of phosphorus in samples of lecithin. However, the presence of calcium in the sample can result in the formation of CaC2, resulting from interaction with the graphite furnace; or in the formation of CaO, which can accumulate on the surface of the lens and graphite furnace equipment (Hughes, Grégoire, Hirohito & Chakrabarti, 1997). The formation of MgO can also occur, but only when low temperatures of vaporization are used. Given this, magnesium was selected as chemical modifier for the proposed method.
3.3. Analytical performance and application Calibration curves were obtained with the use of aqueous standards and by addition of analytes in samples of soybean lecithin diluted in MIBK. In addition to the calibration curve at 213.561 nm, curves obtained with the sum of the analytical signals of the lines located around 213 nm (213.526, 213.617, 213.637 and 213.561 nm) were also evaluated, employing the same thermal conditions as established individually at 213.561 nm. The slopes of the calibration curves are presented in Table 2. Comparing the slopes of the calibration curves, it can be seen that the proposed method can be conducted using external calibration with aqueous standards. This is an important benefit, as the use of external calibration increases the practicality and consistency of the proposed method. Limits of detection (LOQ) of the proposed method were calculated as three times the standard deviation of ten blank measurements, divided by the 11
slope of the calibration curve. The sample dilution was considered in the calculation of the LOD. The precision was expressed in terms of the relative standard deviation (RSD). Table 2 presents the analytical parameters for the proposed method. The sensitivity and precision were better when the sum of the signals of the lines located around 213 nm was used. This resulted in lower values for the limit of detection, in accordance with previous reports (Bechlin, Gomes Neto & Nóbrega, 2013; Resano & García-Ruiz, 2011). The proposed method was applied to the determination of phospholipids in six samples of soybean lecithin and the results obtained were compared with those obtained by ICP OES after sample digestion (Table 3). A paired t test did not show significant differences between the obtained results (95 % confidence level). The concentration of phospholipids present in soy lecithin is directly proportional to the phosphorous content (Erickson, 1995), and depends on the origin of the soybeans as well the conditions used in the oil degumming process (Rydhag & Wilton, 1981). The proportionality factor is calculated based on the average molar mass of phospholipids present in the lecithin divided by the atomic mass of phosphorus. The methods of the American Oil Chemists' Society (AOCS) give a factor of 30 for the estimation of the concentration of phospholipids present in lecithin extracted from crude soybean oil (List, Heakin, Evans, Black & Mounts, 1978; Weihrauch & Son, 1983). The results, expressed as a percentage of phospholipids, are also shown in Table 3 and are in agreement with results obtained previously, where HPLC (28.9 to 44.1%) (Hurst & Martin, 1984), NMR-P (47% and 50.7%) (Nieuwenhuyzen & Tomás, 2008;
12
Helmerick & Koehler, 2003) and EASI-MS (50.4%) (Fernandes, Alberici, Pereira, Cabral, Eberlin & Barrera-Arellano, 2012) were used.
13
4. Conclusion The proposed method allowed the determination of phospholipids in soybean lecithin samples by the determination of P via molecular absorption of PO using HR-CS GF MAS. The analysis was favored by the presence of magnesium as a chemical modifier and presented the advantage of allowing the use of external calibration. When samples were diluted with MIBK, there was no matrix effect that interfered in the analytical signal concerning the PO molecule. Analytical parameters such as limits of detection, precision and accuracy were adequate for the purpose of the work. It is worth mentioning that the sum of lines proved to be effective in improving these parameters, compared with those obtained by the integrated absorbance resulting from the monitoring of a single line.
Acknowledgments The authors are grateful to Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing grants, fellowships and financial support.
Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
14
5. References Almeida, J. S., Brandão, G. C., Santos, G. L., Teixeira, L. S. G. (2016). Fast sequential determination of manganese and chromium in vegetable oil and biodiesel samples by high-resolution continuum source graphite furnace atomic absorption spectrometry. Anal. Methods, 8, 3249–3254. Amorim, F. A. C., Welz, B., Costa, A. C. S., Lepri, F. G., Vale, M. G. R., & Ferreira, S. L. C. (2007). Determination of vanadium in petroleum and petroleum products using atomic spectrometric techniques. Talanta, 72, 349–359. Bechlin, M. A., Gomes Neto, J. A., & Nóbrega, J. A. (2013). Evaluation of lines of boron, phosphorus and sulfur by high-resolution continuum source flame atomic absorption spectrometry for plant analysis. Microchemical Journal, 109, 134–138. Cooper, L., & Ainscough, T. (2016). FT-NIR analysis of lecithin. Inform, 27, 706–707. Dessuy, B. M. (2007). Investigação do comportamento de modificadores químicos para fósforo em forno de grafite usando espectrometria de absorção atômica de linha de fonte contínua de alta resolução. 2007. 88 f. Dissertação (Mestrado) - Instituto de Química, Universidade Federal do Rio Grande
do
Sul,
Porto
Alegre.
URL
http://www.lume.ufrgs.br/handle/10183/11847/. Accessed 26.09.16.
15
Erickson, D. R. (1995). Degumming and Lecithin Processing and Utilization. In Erickson, D. R. (ed), Practical Handbook of Soybean Processing and Utilization (pp. 174–183). Champaign: AOCS Press. Fernandes, G. D., Alberici, R. M., Pereira, G. G., Cabral, E. C., Eberlin, M. N., & Barrera-Arellano, D. (2012). Direct characterization of commercial lecithins by easy ambient sonic-spray ionization mass spectrometry. Food Chemistry, 135, 1855–1860. Gonzalez, M. C., Rodriguez, A. R., & Gonzalez, V. (1987). Determination of vanadium, nickel, iron, copper, and lead in petroleum fractions by atomic absorption spectrophotometry with a graphite furnace. Microchemical Journal, 35, 94–106. Heitmann, U., Becker-Ross, H., Florek, S., Huang, M. D., & Okruss, M. (2006). Determination of non-metals via molecular absorption using high-resolution continuum
source
absorption
spectrometry
and
graphite
furnace
atomization. Journal of Analytical Atomic Spectrometry, 21, 1314–1320. Helmerich, G., & Koehler, P. (2003). Comparison of methods for the quantitative determination of phospholipids in lecithins and flour improvers. Journal of Agricultural and Food Chemistry, 51, 6645–6651. Helmerick, G., & Koehler, P. (2003). Comparison of methods for the quantitative determination of phospholipids in lecithins and flour improvers. Journal of Agricultural and Food Chemistry, 51, 6645–6651. Huang, M. D., Becker-Ross, H., Florek, S., Heitmann, U., & Okruss, M. (2006). Determination of phosphorus by molecular absorption of phosphorus
16
monoxide using a high-resolution continuum source absorption spectrometer and an air–acetylene flame. Journal of Analytical Atomic Spectrometry, 21, 338–345. Hughes, D. M., Grégoire, D. C., Hirohito, N., & Chakrabarti, C. L. (1997). The vaporization of phosphorus compounds and the use of chemical modifiers for the determination of phosphorus by electrothermal vaporization inductively coupled plasma mass spectrometry. Spectrochimica Acta Part B: Atomic Spectroscopy, 52, 517–529. Hurst, W. J., & Martin, R. A. (1984). The analysis of phospholipids in soy lecithin by HPLC. Journal of the American Oil Chemists' Society, 61, 1462–1463. Jangle, R. D., Magar, V. P., & Thorat, B. N. (2013). Phosphatidylcholine and its purification from raw de-oiled soya lecithin. Separation and Purification Technology, 102, 187–195. John, J. V., Park, H., Lee, H. R., Suh, H., & Kim, I. (2015). Simultaneous extraction of phosphatidylcholine and phosphatidylethanolamine from soybean lecithin. European Journal of Lipid Science and Technology, 117, 1647–1654. Lepri, F. G., Dessuy, M. B., Vale, M. G. R., Borges, D. L. G., Welz, B., & Heitmann, U. (2006). Investigation of chemical modifiers for phosphorus in a graphite furnace using high-resolution continuum source atomic absorption spectrometry. Spectrochimica Acta Part B: Atomic Spectroscopy, 61, 934– 944.
17
Li, H., Goulden, M., Cocciardi, R., & Hughes, J. (2009). Fourier transform near infrared spectroscopy as a quality control tool for the analysis of lecithin and by-products during soybean oil processing. Journal of the American Oil Chemists’ Society, 86, 835–841. List, G. R., Heakin, A. J., Evans, C. D., Black, L. T., & Mounts, T. L. (1978). Factor for converting elemental phosphorus to acetone insolubles in crude soybean oil. Journal of the American Oil Chemists' Society, 55, 511–522. Liu, L., Waters, D. L. E., Rose, T. J., Bao, J., & King, G. J. (2013). Phospholipids in rice: Significance in grain quality and health benefits: A review. Food Chemistry, 139, 1133–1145. Martín-Polvillo, M., Albi, T., & Guinda, A. (1994). Determination of trace elements in edible vegetable oils by atomic absorption spectrophotometry. Journal of the American Oil Chemists' Society, 71, 347–353. Mertins, O., Sebben, M., Schneider, P. H., Pohlmann, A. R., & Silveira, N. P. (2008). Caracterização da pureza de fosfatidilcolina da soja através de RMN de 1H e de 31P. Química Nova, 31, 1856–1859. Nieuwenhuyzen, W. V., & Tomás, M. C. (2008). Update on vegetable lecithin and phospholipid technologies. European Journal of Lipid Science and Technology, 110, 472–486. Resano, M., & García-Ruiz, E. (2011). High-resolution continuum source graphite furnace atomic absorption spectrometry: Is it as good as it sounds? A critical review. Analytical and Bioanalytical Chemistry, 399, 323–330.
18
Resano, M., Belarra, M. A., Castillo, J. R., & Vanhaecke, F. (2000). Direct determination of phosphorus in two different plastic materials (PET and PP) by solid sampling-graphite furnace atomic absorption spectrometry. Journal of Analytical Atomic Spectrometry, 15, 1383–1388. Resano, M., Briceño, J., & Belarra, M. A. (2009). Direct determination of phosphorus in biological samples using a solid sampling-high resolutioncontinuum source electrothermal spectrometer: comparison of atomic and molecular
absorption
spectrometry.
Journal
of
Analytical
Atomic
Spectrometry, 24, 1343–1354. Rydhag, L., & Wilton, I. (1981). The function of phospholipids of soybean lecithin in emulsions. Journal of the American Oil Chemists’ Society, 58, 830–837. Szydłowska-Czerniak, A. (2007). MIR spectroscopy and partial least-squares regression for determination of phospholipids in rapeseed oils at various stages of technological process. Food Chemistry, 105, 1179–1187. Totani, Y., Pretorius, H. E., & Plessis, L. M. (1982). Extraction of phospholipids from plant oils and colorimetric determination of total phosphorus. Journal of the American Oil Chemists' Society, 59, 162–163. Vale, M. G. R., Damin, I. C. F., Klassen, A., Silva, M. M., Welz, B., Silva, A. F., Lepri, F. G., Borges, D. L. G., & Heitmann, U. (2004). Method development for the determination of nickel in petroleum using line-source and highresolution
continuum-source
graphite
furnace
atomic
absorption
spectrometry. Microchemical Journal, 77, 131–140.
19
Weihrauch, J. L., & Son, Y. S. (1983). Phospholipid content of foods. Journal of the American Oil Chemists' Society, 60, 1971–1978. Welz, B. High-resolution continuum source AAS: the better way to perform atomic absorption spectrometry. (2005). Analytical and Bioanalytical Chemistry, 381, 69–71. Welz, B., Lepri, F. G., Araujo, R. G. O., Ferreira, S. L. C., Huang, M. D., Okruss, M., & Becker-Ross, H. (2009). Determination of phosphorus, sulfur and the halogens using high-temperature molecular absorption spectrometry in flames and furnaces - A review. Analytica Chimica Acta, 647, 137–148.
20
Figure caption
Fig. 1. Pyrolysis and vaporization curves for the determination of P in soy lecithin via PO molecular absorption in the absence (●) and in the presence of Ca (■) and Mg (▲) at (A) 213.561 nm, (B) 246.400 nm and (C) 324.620 nm (n = 3).
21
Table 1 Temperature program for the determination of phosphorus in soybean lecithin samples using HR-CS GF MAS via molecular absorption of phosphorus monoxide. Stage
Temperature, °C
Ramp, °C s-1
Hold time, s
Drying
110
3
20
2.0
Drying
140
5
15
2.0
Pyrolysis
1300
200
20
2.0
Autozero
1300
0
5
0
Vaporization
2300
2500
5
0
Clean
2450
500
4
2.0
Gas flow rate, L min-1
22
Table 2 Analytical parameters obtained for the determination of phosphorus via molecular absorption of PO using HR-CS GF MAS. Line
Slope 1
Slope 2
LOD, mg g-1
RSD, %
sum of signalsa
0.0051 ± 0.0004 (r= 0.9988)
0.0043 ± 0.0005 (r= 0.9976)
2.35
2.5
213.561 nm
0.0014 ± 0.0002 (r= 0.9982)
0.0012 ± 0.0001 (r= 0.9980)
11.4
4.2
a
Sum of analytical signals of lines located around 213 nm (213.526, 213.617, 213.637 and 213.561 nm). Slope 1: angular coefficient of the calibration curve obtained by aqueous media; Slope 2: angular coefficient of the calibration curve obtained by addition of analyte in a sample of soybean lecithin diluted in MIBK; LOD: limit of detection; RSD: relative standard deviation (n= 10, 2.2% of phosphorus).
23
Table 3 Determination of phosphorus and phospholipids in soybean lecithin samples.
Sample
Phosphorus, %
Phospholipids, %
HR-CS GF MAS
ICP OES
HR-CS GF MAS
ICP OES
1
1.29 ± 0.01
1.45 ± 0.01
38.7 ± 0.1
43.5 ± 0.3
2
1.35 ± 0.03
1.22 ± 0.06
40 ± 1
37 ± 2
3
1.51 ± 0.05
1.65 ± 0.02
45 ± 2
49.5 ± 0.5
4
1.35 ± 0.09
1.43 ± 0.02
40 ± 3
42.9 ± 0.6
5
1.44 ± 0.04
1.38 ± 0.01
43 ± 1
41.4 ± 0.1
6
1.27 ± 0.06
1.23 ± 0.03
38.1 ± 0.2
37 ± 1
24
Fig. 1
25
Highlights HR-CS GF MAS was employed for indirect determination of phospholipids Soybean lecithin samples were analyzed Phosphorus was determined via molecular absorption of phosphorus monoxide Simple sample preparation by dilution with MIBK
26
S0308-8146(17)30019-5 http://dx.doi.org/10.1016/j.foodchem.2017.01.019 FOCH 20410
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
26 September 2016 3 January 2017 4 January 2017
Please cite this article as: Pires, L.N., Brandão, G.C., Teixeira, L.S.G., Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.01.019
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry
Laís N. Pires a, Geovani C. Brandãoa,b, Leonardo S. G. Teixeiraa,b*
a
Universidade Federal da Bahia, Instituto de Química, Departamento de
Química Analítica, Campus Universitário de Ondina, 40170-115, Salvador, Bahia, Brasil. b
INCT de Energia e Ambiente - Universidade Federal da Bahia, Instituto de
Química, Campus Universitário de Ondina, 40170-115, Salvador, Bahia, Brasil.
_________________________________________________________________________ *
Corresponding author: e-mail: [email protected]; Tel + 55 71 3283-6830; Fax +55 71 3235-5166
1
Determination of phospholipids in soybean lecithin samples via the phosphorus monoxide molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry
Abstract This paper presents a method for determining phospholipids in soybean lecithin samples by phosphorus determination using high-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS GF MAS) via molecular absorption of phosphorus monoxide. Samples were diluted in methyl isobutyl ketone. The best conditions were found to be 213.561 nm with a pyrolysis temperature of 1300°C, a volatilization temperature of 2300°C and Mg as a chemical modifier. To increase the analytical sensitivity, measurement of the absorbance signal was obtained by summing molecular transition lines for PO surrounding 213 nm: 213.561, 213.526, 213.617 and 213.637 nm. The limit of detection was 2.35 mg g-1 and the precision, evaluated as relative standard deviation (RSD), was 2.47% (n=10) for a sample containing 2.2% (w/v) phosphorus. The developed method was applied for the analysis of commercial samples of soybean lecithin. The determined concentrations of phospholipids in the samples varied between 38.1 and 45% (w/v).
Keywords:
phospholipids;
soybean
lecithin;
phosphorus;
phosphorus
monoxide; HR-CS GF MAS.
2
1. Introduction Lecithin is a mixture found in animal tissues and in vegetable oils, being composed of choline, glycerol, fatty acids, glycolipids, triacylglycerols and, mainly, phospholipids such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, with smaller quantities of phosphatidic acid and phosphatidylserine (Cooper & Ainscough, 2016). Among all vegetable oils, soybean oil contains the greatest amount of lecithin, which is separated from the crude oil by the degumming process. Crude lecithin is a viscous liquid with a color ranging from brown to pale yellow. It contains about 60–70% phospholipids, 30–40% of neutral oil and about 0.5–2% moisture (Rydhag & Wilton, 1981). The phospholipids present in lecithin are essential components of cell membranes and, therefore, essential for the growth, maturation and proper function of cells (Liu, Waters, Rose, Bao & King, 2013). Phosphatidylcholine is present in the greatest quantity in lecithin and makes a direct contribution to the formation of bilipid layers in cell membranes. It is also a precursor of choline synthesis within the body, a nutrient that is essential for the transport of fats and a precursor of acetylcholine — itself an important neurotransmitter necessary for memory and other brain functions (Jangle, Magar & Thorat, 2013). Phospholipids have gained special attention because of some intrinsic health benefits. Studies have suggested the therapeutic use of phospholipids such as lecithin for the improvement of memory, liver protection, cholesterol reduction and treating neurological disorders (Szydłowska-Czerniak, 2007). Furthermore, due to their amphiphilic nature, availability and excellent functionality, phospholipids have wide application as an emulsifier in the food, 3
pharmaceutical and cosmetic industries (Jangle, Magar & Thorat, 2013; Mertins, Sebben, Schneider, Pohlmann & Silveira, 2008). Thus, there is a necessity for analytical methods to monitor the quality of commercial soybean lecithin, in particular related to their phospholipid content. Due to its complex composition, most reports on the analysis of lecithin and fractionation of its phospholipids involve expensive and laborious procedures (Fernandes, Alberici, Pereira, Cabral, Eberlin & Barrera-Arellano, 2012). Various analytical techniques have been used for the determination of phospholipids in soybean lecithin, for example, easy ambient sonic-spray ionization mass spectrometry (EASI-MS) spectroscopy (Fernandes, Alberici, Pereira, Cabral, Eberlin & Barrera-Arellano, 2012), Fourier transform near infrared spectroscopy (FT-NIR) (Li, Goulden, Cocciardi, Hughes, 2009), thinlayer chromatography (TLC) (Helmerich & Koehler, 2003), high-performance liquid chromatography (HPLC) (Helmerich &
Koehler, 2003), (31)P nuclear
magnetic resonance spectroscopy ((31)P NMR) (Helmerich & Koehler, 2003), UV-vis spectroscopy (John, Park, Lee, Suh & Kim, 2015) and colorimetry (Totani, Pretorius & Plessis, 1982). Although phosphorus determination in complex matrices using atomic spectrometric techniques is still challenging, high-resolution continuum source molecular absorption spectrometry (HR-CS MAS) has shown promise for overcoming these difficulties. Molecular absorption spectrometry (MAS) is a technique developed for monitoring the absorption of diatomic molecules such as NO, CS and PO (Welz, Lepri, Araujo, Ferreira, Huang, Okruss & Becker-Ross, 2009). The technique has been employed in the determination of phosphorus via the PO molecule in wheat flour samples (Heitmann, Becker-Ross, Florek, Huang & Okruss, 2006)
4
and biological materials (Resano, Briceño & Belarra, 2009). However, it should be noted that the determination of phospholipids in soybean lecithin samples via the PO molecule by high-resolution continuum source graphite furnace molecular absorption spectrometry (HR-CS GF MAS) has not yet been recorded in the literature. In this study, an analytical method is proposed for the determination of phospholipids in soybean lecithin samples by HR-CS GF MAS via the molecular absorption of phosphorus monoxide. Different organic solvents were tested as diluents to allow the introduction of samples into the spectrometer. Moreover, the temperature conditions for phosphorus determination by PO absorption were studied for different transition lines in the presence and absence of chemical modifiers.
2. Experimental 2.1. Instrumentation All measurements of molecular absorption of PO were performed employing a high-resolution continuum source electrothermal atomic absorption spectrometer model ContrAA 700 (Analytik Jena AG, Jena, Germany). The spectrometer was equipped with a xenon short-arc lamp as a continuum radiation source, a double-echelle monochromator and a charge-coupled device (CCD) array detector. Pyrolytically coated graphite tubes and platforms, provided by the same manufacturer, were transversely heated and used for all determinations. Argon with a purity of 99.999% (White Martins, São Paulo, Brazil) was used as the purge gas at a flow rate of 2.0 L min-1. The integrated
5
peak area using three pixels was employed as the analytical signal. The temperature program used for the determinations is presented in Table 1. As a comparative procedure, the soybean lecithin samples were subjected to acid digestion employing a heating block model TE-040/25 (Tecnal, São Paulo, Brazil). After digestion, an inductively coupled plasma optical emission spectrometry (ICP OES) instrument, model 720 series (Agilent Technologies, Santa Clara, USA) was used to determine the phosphorus content at 213.618 nm. The equipment was operated under the manufacturer’s recommended conditions for power (1.10 kW), plasma gas flow (15 L min-1), auxiliary gas flow (1.5 L min-1) and nebulizer gas flow (0.75 L min-1), using a Sturman-Master chamber and a V-Groove nebulizer.
2.2. Reagents, solutions and samples All chemicals used in the experiments were of analytical grade. The aqueous solutions were prepared using ultrapure water (resistivity > 18 MΩ cm) obtained from a Milli-Q® purification system (Millipore, Bedford, USA). All flasks and glassware were soaked in 10% (v/v) HNO3 for at least 12 h and rinsed with water before use. A 0.1% (w/v) Ca solution and a 0.1% (w/v) Mg solution, used as chemical modifiers, were prepared by dissolving CaCl2 (Merck, Darmstadt, Germany) and Mg(NO3)2 (Merck), respectively, in propan-1-ol (Vetec, Rio de Janeiro, Brazil). A 1000 mg L-1 phosphorus stock solution was prepared by dissolving anhydrous monobasic ammonium phosphate (Merck) in water. Methyl isobutyl ketone (Merck), pentane (Merck), hexane (Merck), acetone (Vetec), acetic acid (Vetec), methylethylketone (Merck), propan-1-ol
6
(Vetec) and methanol (Vetec) were tested as solvents for the dilution of the soybean lecithin samples. The proposed procedure was applied to the analysis of six soybean lecithin samples purchased in markets in Salvador City, Bahia, Brazil.
2.3. Sample preparation Approximately 0.1000 g of each soybean lecithin sample was weighed directly into a polyethylene tube and diluted in 10 mL of MIBK for use in HR-CS GF MAS. All samples were prepared in triplicate. The results obtained with the proposed method were compared with those resulting from samples treated by acid digestion and determination by ICP OES. For ICP OES, approximately 0.2000 g of each sample was weighed directly into a digester. Then, 3.0 mL of concentrated HNO3 and 1.0 mL of concentrated H2SO4 were added. The system temperature was varied in steps: in the first 15 min, the temperature was maintained at 150 °C and then maintained at 180 °C for another 15 min. Afterwards, 2.0 mL of H2O2 30% (v/v) was added and the temperature was raised to 220 °C. The total time of the procedure was 2 h. After cooling to room temperature, the digestion was diluted with water for 10 mL in polyethylene bottles. The digestion of all samples was performed in triplicate and analytical blanks were also obtained in the same way. Working standard solutions for phosphorus were prepared by dilution from 1000 mg L-1 stock solutions (Specsol Quimlab, Brazil).
7
3. Results and discussion 3.1. Sample dilution Aspiration of high viscosity samples into the detection system can be a problem when graphite furnace atomic absorption spectrometry (GF AAS) is employed in elemental determination (Almeida, Brandão, Santos & Teixeira, 2016). Dilution in organic solvents is a method of sample preparation that allows adjustment of the viscosity of the sample, enabling simple and fast analysis of oily matrices by atomic spectrometric techniques. Among the most frequently used solvents for oily sample dilution are MIBK, pentane, hexane, acetone, acetic acid, methylethylketone, propan-1-ol, methanol and toluene (Amorim, Welz, Costa, Lepri, Vale & Ferreira, 2007; Vale, Damin, Klassen, Silva, Welz, Silva, Lepri, Borges & Heitmann, 2004). Because lecithin is composed of phospholipids with different groups that impart different polarity and solubility in organic solvents (John, Park, Lee, Suh & Kim, 2015), several of these solvents were tested for the dilution of lecithin samples. Soybean lecithin samples were partially dissolved in acetone, methyl ethyl ketone and acetic acid. Appropriate solubilization was also not observed when the propane and methanol were used. However, the samples were completely dissolved when MIBK, pentane and hexane were used as diluents. A study of the values and stability of analytical signals was then accomplished by observing the profile of absorption peaks obtained with the use of these three solvents. Stable analytical signals were found for the three solvents after successive measurements. Additionally, the analytical responses were similar when MIBK, pentane or cyclohexane were used; however, narrower peaks and lower background signals were obtained with samples diluted in MIBK. Thus, 8
MIBK was used as the solvent for the dilution of lecithin samples, in agreement with previous work that used this solvent for the dilution of organic matrices for determination by GF AAS (Vale, Damin, Klassen, Silva, Welz, Silva, Lepri, Borges & Heitmann, 2004; Gonzalez, Rodriguez & Gonzalez, 1987; MartínPolvillo, Albi & Guinda, 1994).
3.2. Optimization of the furnace heating program The diatomic PO molecule presents three different electronic transitions and, consequently, has a large number of molecular lines with different sensitivities that can be used for the determination of phosphorus (Welz, 2005). Huang et al. (2006), for example, studied the absorption of this molecule at 246.40, 247.78, 324.62 and 327.04 nm, with attention to possible interference in each line, while Resano et al. (2009) have conducted studies on the absorption of PO exploring the sum of the lines in the spectral region around 213.618 nm. In this work, studies were performed to define the best electrothermal conditions and the best chemical modifier to be used for the lines of molecular absorption of PO at 213.561, 246.400 and 324.620 nm, in order to establish the most appropriate line for the quantification of phospholipids in soybean lecithin samples. Modifiers that do not favor P reduction and atomization were used, because otherwise an spectral interference would appear. A soybean lecithin sample diluted in MIBK was employed, varying pyrolysis and vaporization temperatures between 700-1800 °C and 1700-2700 °C, respectively, in the absence and in the presence of calcium and magnesium as chemical modifiers instead of more traditional modifiers like Pd, which will produce atomic P (Lepri, 9
Dessuy, Vale, Borges, Welz & Heitmann, 2006; Huang, 2006; Resano, 2009; Resano, Belarra, Castillo & Vanhaecke, 2000). The pyrolysis and vaporization curves obtained for each line are presented in Figure 1. The different sensitivities of each absorption line were confirmed with the pyrolysis and vaporization curves, wherein the highest analytical signals at 213.561 nm were obtained in the presence of calcium, while the highest analytical signals at 246.400 and 324.620 nm were obtained in the presence of magnesium. In addition, the results obtained showed that the pyrolysis and vaporization curves were well defined only at 213.561 nm, with analytical signals favored by the presence of the modifiers. These results are consistent with previous work, where difficulties in the optimization of pyrolysis and vaporization temperatures have been reported using the molecular transition lines at 246.400 and 324.620 nm (Dessuy, 2007). A further observation was that vaporization temperatures over 2300 °C can result in a decrease in sensitivity for the determination of PO, which could be the result of factors such as: formation of atomic phosphorus, which begins to be significant at higher temperatures; or the possible formation of other molecular species such as P2 and PO2 (Resano, Briceño & Belarra, 2009). This behavior was observed in the study of temperature of vaporization at 213.561 nm in the presence of the modifiers, where the absorbance signals decreased slightly at temperatures up to 2300 °C. However, these factors did not impair the determination when temperatures up to 2300 °C were employed in the presence of the modifiers. The pyrolysis and vaporization curves at 213.561 nm also showed that the thermal behavior was similar when calcium and magnesium were used as
10
chemical modifiers. Probably the same mechanism of stabilization of the analyte was obtained for both modifiers, which indicates that either of them could be used for the determination of phosphorus in samples of lecithin. However, the presence of calcium in the sample can result in the formation of CaC2, resulting from interaction with the graphite furnace; or in the formation of CaO, which can accumulate on the surface of the lens and graphite furnace equipment (Hughes, Grégoire, Hirohito & Chakrabarti, 1997). The formation of MgO can also occur, but only when low temperatures of vaporization are used. Given this, magnesium was selected as chemical modifier for the proposed method.
3.3. Analytical performance and application Calibration curves were obtained with the use of aqueous standards and by addition of analytes in samples of soybean lecithin diluted in MIBK. In addition to the calibration curve at 213.561 nm, curves obtained with the sum of the analytical signals of the lines located around 213 nm (213.526, 213.617, 213.637 and 213.561 nm) were also evaluated, employing the same thermal conditions as established individually at 213.561 nm. The slopes of the calibration curves are presented in Table 2. Comparing the slopes of the calibration curves, it can be seen that the proposed method can be conducted using external calibration with aqueous standards. This is an important benefit, as the use of external calibration increases the practicality and consistency of the proposed method. Limits of detection (LOQ) of the proposed method were calculated as three times the standard deviation of ten blank measurements, divided by the 11
slope of the calibration curve. The sample dilution was considered in the calculation of the LOD. The precision was expressed in terms of the relative standard deviation (RSD). Table 2 presents the analytical parameters for the proposed method. The sensitivity and precision were better when the sum of the signals of the lines located around 213 nm was used. This resulted in lower values for the limit of detection, in accordance with previous reports (Bechlin, Gomes Neto & Nóbrega, 2013; Resano & García-Ruiz, 2011). The proposed method was applied to the determination of phospholipids in six samples of soybean lecithin and the results obtained were compared with those obtained by ICP OES after sample digestion (Table 3). A paired t test did not show significant differences between the obtained results (95 % confidence level). The concentration of phospholipids present in soy lecithin is directly proportional to the phosphorous content (Erickson, 1995), and depends on the origin of the soybeans as well the conditions used in the oil degumming process (Rydhag & Wilton, 1981). The proportionality factor is calculated based on the average molar mass of phospholipids present in the lecithin divided by the atomic mass of phosphorus. The methods of the American Oil Chemists' Society (AOCS) give a factor of 30 for the estimation of the concentration of phospholipids present in lecithin extracted from crude soybean oil (List, Heakin, Evans, Black & Mounts, 1978; Weihrauch & Son, 1983). The results, expressed as a percentage of phospholipids, are also shown in Table 3 and are in agreement with results obtained previously, where HPLC (28.9 to 44.1%) (Hurst & Martin, 1984), NMR-P (47% and 50.7%) (Nieuwenhuyzen & Tomás, 2008;
12
Helmerick & Koehler, 2003) and EASI-MS (50.4%) (Fernandes, Alberici, Pereira, Cabral, Eberlin & Barrera-Arellano, 2012) were used.
13
4. Conclusion The proposed method allowed the determination of phospholipids in soybean lecithin samples by the determination of P via molecular absorption of PO using HR-CS GF MAS. The analysis was favored by the presence of magnesium as a chemical modifier and presented the advantage of allowing the use of external calibration. When samples were diluted with MIBK, there was no matrix effect that interfered in the analytical signal concerning the PO molecule. Analytical parameters such as limits of detection, precision and accuracy were adequate for the purpose of the work. It is worth mentioning that the sum of lines proved to be effective in improving these parameters, compared with those obtained by the integrated absorbance resulting from the monitoring of a single line.
Acknowledgments The authors are grateful to Fundação de Amparo a Pesquisa do Estado da Bahia (FAPESB), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing grants, fellowships and financial support.
Conflict of Interests The authors declare that there is no conflict of interests regarding the publication of this paper.
14
5. References Almeida, J. S., Brandão, G. C., Santos, G. L., Teixeira, L. S. G. (2016). Fast sequential determination of manganese and chromium in vegetable oil and biodiesel samples by high-resolution continuum source graphite furnace atomic absorption spectrometry. Anal. Methods, 8, 3249–3254. Amorim, F. A. C., Welz, B., Costa, A. C. S., Lepri, F. G., Vale, M. G. R., & Ferreira, S. L. C. (2007). Determination of vanadium in petroleum and petroleum products using atomic spectrometric techniques. Talanta, 72, 349–359. Bechlin, M. A., Gomes Neto, J. A., & Nóbrega, J. A. (2013). Evaluation of lines of boron, phosphorus and sulfur by high-resolution continuum source flame atomic absorption spectrometry for plant analysis. Microchemical Journal, 109, 134–138. Cooper, L., & Ainscough, T. (2016). FT-NIR analysis of lecithin. Inform, 27, 706–707. Dessuy, B. M. (2007). Investigação do comportamento de modificadores químicos para fósforo em forno de grafite usando espectrometria de absorção atômica de linha de fonte contínua de alta resolução. 2007. 88 f. Dissertação (Mestrado) - Instituto de Química, Universidade Federal do Rio Grande
do
Sul,
Porto
Alegre.
URL
http://www.lume.ufrgs.br/handle/10183/11847/. Accessed 26.09.16.
15
Erickson, D. R. (1995). Degumming and Lecithin Processing and Utilization. In Erickson, D. R. (ed), Practical Handbook of Soybean Processing and Utilization (pp. 174–183). Champaign: AOCS Press. Fernandes, G. D., Alberici, R. M., Pereira, G. G., Cabral, E. C., Eberlin, M. N., & Barrera-Arellano, D. (2012). Direct characterization of commercial lecithins by easy ambient sonic-spray ionization mass spectrometry. Food Chemistry, 135, 1855–1860. Gonzalez, M. C., Rodriguez, A. R., & Gonzalez, V. (1987). Determination of vanadium, nickel, iron, copper, and lead in petroleum fractions by atomic absorption spectrophotometry with a graphite furnace. Microchemical Journal, 35, 94–106. Heitmann, U., Becker-Ross, H., Florek, S., Huang, M. D., & Okruss, M. (2006). Determination of non-metals via molecular absorption using high-resolution continuum
source
absorption
spectrometry
and
graphite
furnace
atomization. Journal of Analytical Atomic Spectrometry, 21, 1314–1320. Helmerich, G., & Koehler, P. (2003). Comparison of methods for the quantitative determination of phospholipids in lecithins and flour improvers. Journal of Agricultural and Food Chemistry, 51, 6645–6651. Helmerick, G., & Koehler, P. (2003). Comparison of methods for the quantitative determination of phospholipids in lecithins and flour improvers. Journal of Agricultural and Food Chemistry, 51, 6645–6651. Huang, M. D., Becker-Ross, H., Florek, S., Heitmann, U., & Okruss, M. (2006). Determination of phosphorus by molecular absorption of phosphorus
16
monoxide using a high-resolution continuum source absorption spectrometer and an air–acetylene flame. Journal of Analytical Atomic Spectrometry, 21, 338–345. Hughes, D. M., Grégoire, D. C., Hirohito, N., & Chakrabarti, C. L. (1997). The vaporization of phosphorus compounds and the use of chemical modifiers for the determination of phosphorus by electrothermal vaporization inductively coupled plasma mass spectrometry. Spectrochimica Acta Part B: Atomic Spectroscopy, 52, 517–529. Hurst, W. J., & Martin, R. A. (1984). The analysis of phospholipids in soy lecithin by HPLC. Journal of the American Oil Chemists' Society, 61, 1462–1463. Jangle, R. D., Magar, V. P., & Thorat, B. N. (2013). Phosphatidylcholine and its purification from raw de-oiled soya lecithin. Separation and Purification Technology, 102, 187–195. John, J. V., Park, H., Lee, H. R., Suh, H., & Kim, I. (2015). Simultaneous extraction of phosphatidylcholine and phosphatidylethanolamine from soybean lecithin. European Journal of Lipid Science and Technology, 117, 1647–1654. Lepri, F. G., Dessuy, M. B., Vale, M. G. R., Borges, D. L. G., Welz, B., & Heitmann, U. (2006). Investigation of chemical modifiers for phosphorus in a graphite furnace using high-resolution continuum source atomic absorption spectrometry. Spectrochimica Acta Part B: Atomic Spectroscopy, 61, 934– 944.
17
Li, H., Goulden, M., Cocciardi, R., & Hughes, J. (2009). Fourier transform near infrared spectroscopy as a quality control tool for the analysis of lecithin and by-products during soybean oil processing. Journal of the American Oil Chemists’ Society, 86, 835–841. List, G. R., Heakin, A. J., Evans, C. D., Black, L. T., & Mounts, T. L. (1978). Factor for converting elemental phosphorus to acetone insolubles in crude soybean oil. Journal of the American Oil Chemists' Society, 55, 511–522. Liu, L., Waters, D. L. E., Rose, T. J., Bao, J., & King, G. J. (2013). Phospholipids in rice: Significance in grain quality and health benefits: A review. Food Chemistry, 139, 1133–1145. Martín-Polvillo, M., Albi, T., & Guinda, A. (1994). Determination of trace elements in edible vegetable oils by atomic absorption spectrophotometry. Journal of the American Oil Chemists' Society, 71, 347–353. Mertins, O., Sebben, M., Schneider, P. H., Pohlmann, A. R., & Silveira, N. P. (2008). Caracterização da pureza de fosfatidilcolina da soja através de RMN de 1H e de 31P. Química Nova, 31, 1856–1859. Nieuwenhuyzen, W. V., & Tomás, M. C. (2008). Update on vegetable lecithin and phospholipid technologies. European Journal of Lipid Science and Technology, 110, 472–486. Resano, M., & García-Ruiz, E. (2011). High-resolution continuum source graphite furnace atomic absorption spectrometry: Is it as good as it sounds? A critical review. Analytical and Bioanalytical Chemistry, 399, 323–330.
18
Resano, M., Belarra, M. A., Castillo, J. R., & Vanhaecke, F. (2000). Direct determination of phosphorus in two different plastic materials (PET and PP) by solid sampling-graphite furnace atomic absorption spectrometry. Journal of Analytical Atomic Spectrometry, 15, 1383–1388. Resano, M., Briceño, J., & Belarra, M. A. (2009). Direct determination of phosphorus in biological samples using a solid sampling-high resolutioncontinuum source electrothermal spectrometer: comparison of atomic and molecular
absorption
spectrometry.
Journal
of
Analytical
Atomic
Spectrometry, 24, 1343–1354. Rydhag, L., & Wilton, I. (1981). The function of phospholipids of soybean lecithin in emulsions. Journal of the American Oil Chemists’ Society, 58, 830–837. Szydłowska-Czerniak, A. (2007). MIR spectroscopy and partial least-squares regression for determination of phospholipids in rapeseed oils at various stages of technological process. Food Chemistry, 105, 1179–1187. Totani, Y., Pretorius, H. E., & Plessis, L. M. (1982). Extraction of phospholipids from plant oils and colorimetric determination of total phosphorus. Journal of the American Oil Chemists' Society, 59, 162–163. Vale, M. G. R., Damin, I. C. F., Klassen, A., Silva, M. M., Welz, B., Silva, A. F., Lepri, F. G., Borges, D. L. G., & Heitmann, U. (2004). Method development for the determination of nickel in petroleum using line-source and highresolution
continuum-source
graphite
furnace
atomic
absorption
spectrometry. Microchemical Journal, 77, 131–140.
19
Weihrauch, J. L., & Son, Y. S. (1983). Phospholipid content of foods. Journal of the American Oil Chemists' Society, 60, 1971–1978. Welz, B. High-resolution continuum source AAS: the better way to perform atomic absorption spectrometry. (2005). Analytical and Bioanalytical Chemistry, 381, 69–71. Welz, B., Lepri, F. G., Araujo, R. G. O., Ferreira, S. L. C., Huang, M. D., Okruss, M., & Becker-Ross, H. (2009). Determination of phosphorus, sulfur and the halogens using high-temperature molecular absorption spectrometry in flames and furnaces - A review. Analytica Chimica Acta, 647, 137–148.
20
Figure caption
Fig. 1. Pyrolysis and vaporization curves for the determination of P in soy lecithin via PO molecular absorption in the absence (●) and in the presence of Ca (■) and Mg (▲) at (A) 213.561 nm, (B) 246.400 nm and (C) 324.620 nm (n = 3).
21
Table 1 Temperature program for the determination of phosphorus in soybean lecithin samples using HR-CS GF MAS via molecular absorption of phosphorus monoxide. Stage
Temperature, °C
Ramp, °C s-1
Hold time, s
Drying
110
3
20
2.0
Drying
140
5
15
2.0
Pyrolysis
1300
200
20
2.0
Autozero
1300
0
5
0
Vaporization
2300
2500
5
0
Clean
2450
500
4
2.0
Gas flow rate, L min-1
22
Table 2 Analytical parameters obtained for the determination of phosphorus via molecular absorption of PO using HR-CS GF MAS. Line
Slope 1
Slope 2
LOD, mg g-1
RSD, %
sum of signalsa
0.0051 ± 0.0004 (r= 0.9988)
0.0043 ± 0.0005 (r= 0.9976)
2.35
2.5
213.561 nm
0.0014 ± 0.0002 (r= 0.9982)
0.0012 ± 0.0001 (r= 0.9980)
11.4
4.2
a
Sum of analytical signals of lines located around 213 nm (213.526, 213.617, 213.637 and 213.561 nm). Slope 1: angular coefficient of the calibration curve obtained by aqueous media; Slope 2: angular coefficient of the calibration curve obtained by addition of analyte in a sample of soybean lecithin diluted in MIBK; LOD: limit of detection; RSD: relative standard deviation (n= 10, 2.2% of phosphorus).
23
Table 3 Determination of phosphorus and phospholipids in soybean lecithin samples.
Sample
Phosphorus, %
Phospholipids, %
HR-CS GF MAS
ICP OES
HR-CS GF MAS
ICP OES
1
1.29 ± 0.01
1.45 ± 0.01
38.7 ± 0.1
43.5 ± 0.3
2
1.35 ± 0.03
1.22 ± 0.06
40 ± 1
37 ± 2
3
1.51 ± 0.05
1.65 ± 0.02
45 ± 2
49.5 ± 0.5
4
1.35 ± 0.09
1.43 ± 0.02
40 ± 3
42.9 ± 0.6
5
1.44 ± 0.04
1.38 ± 0.01
43 ± 1
41.4 ± 0.1
6
1.27 ± 0.06
1.23 ± 0.03
38.1 ± 0.2
37 ± 1
24
Fig. 1
25
Highlights HR-CS GF MAS was employed for indirect determination of phospholipids Soybean lecithin samples were analyzed Phosphorus was determined via molecular absorption of phosphorus monoxide Simple sample preparation by dilution with MIBK
26