- Email: [email protected]
HPLC analysis of organic acids using a novel stationary phase
Talanta 78 (2009) 643–646
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Short communication
HPLC analysis of organic acids using a novel stationary phase A. Rodríguez-Bernaldo de Quirós ∗ , M.A. Lage-Yusty, J. López-Hernández Analytical Chemistry, Nutrition and Bromatology Department, Pharmacy Faculty, Campus Sur s/n, University of Santiago de Compostela, 15782 Santiago de Compostela (La Coru˜ na), Spain
a r t i c l e
i n f o
Article history: Received 1 September 2008 Received in revised form 3 November 2008 Accepted 14 November 2008 Available online 21 November 2008 Keywords: Organic acids Stationary phase Liquid chromatography Mass spectrometry
a b s t r a c t In the present work, a high performance liquid chromatographic method with UV detection for the separation of six organic acids including, tartaric, malic, acetic, lactic, citric and succinic is described. The separation was performed on a novel stationary phase TEKNOKROMA, Tr-010065 Mediterranea sea18 (15 cm × 0.4 cm, i.d. 3 m) and using water with a 0.1% (v/v) of formic acid as mobile phase. The advantages of this packing over a conventional octadecylsilane (ODS2) column are reported. The method was validated with respect to linearity, limits of detection and repeatabilities within day and between days and satisfactory results were obtained. The proposed method was applied for the determination of these compounds in commercially available white wines. The samples were injected directly without previous treatment. LC–MS was used as a confirmatory technique. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The analysis of organic acids in different food items such as fruit juices, vegetables, dairy products, coffee and wine is of great interest for food industry since these compounds are responsible of sensory properties and may also influence their stability [1–5]. Regarding the analytical techniques, simple rapid and reliable methods are required for control purposes. Gas chromatography with flame ionization detector has been used to determine carboxylic acids in citric fruits [6] however this procedure presents a disadvantage, a derivatization step is necessary for non-volatile acids. Liquid chromatography with different separation mechanism including ion-exchange [4,7–9], ion-exclusion [7,10–12] and reversed phase [3,7,13,14] has been widely employed in different matrix. Nowadays capillary zone electrophoresis (CZE) with photodiode array detection appears as a promising analytical tool to determine these compounds in short time [15]. In complex samples mass spectrometry coupled to capillary electrophoresis or liquid chromatography constitutes a powerful technique due to its high sensitivity and selectivity [16–19]. In chromatography, the selection of the stationary phase is essential in order to achieve a suitable separation. In the present work a new reversed packing based on perfectly spherical par-
∗ Corresponding author. Tel.: +34 981 598450; fax: +34 981 594912. E-mail address: [email protected] (A.R.-B. de Quirós). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.11.013
ticles of ultra pure silica and suitable for a wide pH range and aqueous mobile phases was used to analyse six organic acids including, tartaric, malic, lactic, acetic, citric and succinic. A comparison with a conventional octadecylsilane (ODS2) column in what concerns to system suitability parameters such as number of theoretical plates, and peaks width, and sensitivity is reported. The proposed method was validated in terms of linearity, limits of detection and repeatabilities within day and between days. In the second part of the work the method was applied to analyse white wine samples. LC–MS was used as a confirmatory technique. 2. Experimental 2.1. Chemicals and standard solutions All chemicals were of analytical grade. Formic acid (98–100%) was from Riedel-de Haën (Seelze, Germany) and meta-phosphoric acid was purchased from Sigma (St. Louis, MO, USA). Ultrapure water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Organic acid standards were obtained as follows: citric acid (≥99.5%) and dl-lactic acid (∼90%) were from Fluka BioChemika (Steinheim, Germany), malic acid (95–100%), succinic acid (99%) and dl-tartaric acid (99%) were from Sigma–Aldrich (St. Louis, MO, USA) and acetic acid (99–100%) was from Riedel-de Haën (Seelze, Germany). Stock standard solutions were dissolved in mobile phase, Milli-Q water (0.1% formic acid), and stored at 4 ◦ C in the dark.
644
A.R.-B. de Quirós et al. / Talanta 78 (2009) 643–646
Table 1 System suitability parameters and limits of detection of both stationary phases. Compound
Teknokroma ODS2 5 m column a
Width Tartaric Malic Lactic Acetic Citric Succinic a b c
0.20 0.24 0.38 0.21 0.37 0.41
± ± ± ± ± ±
Mediterranea sea18 3 m column b
0.04 0.13 0.09 0.01 0.02 0.01
c
Plates
LoD
Widtha
2281.7 2738.7 1324 4738.8 2335.1 2644.6
0.50 0.70 1.00 1.00 0.70 1.00
0.21 0.15 0.16 0.16 0.23 0.25
± ± ± ± ± ±
0.009 0.005 0.005 0.007 0.007 0.004
Platesb
LoDc
1679.11 5224.7 6500.2 7180.1 10729.9 10300.3
0.10 0.10 0.10 0.10 0.50 0.30
Peak-width (mean of 10 injections). Plates (mean of 10 injections). mg/L.
2.2. Sampling
2.4. Identification and quantification
Commercial available white wines from Galicia (NW Spain) and from the same vintage (2006) were analysed. The samples were filtered through a 50 m PTFE membrane filter (Advantec MFS, INC, CA, USA) and directly injected into the chromatograph without previous treatment. The analyses were performed in triplicate.
Identification of organic acids was made by comparison of their retention times with those of pure standards solutions. Moreover, citric acid, lactic acid, malic acid, succinic acid and tartaric acid were also confirmed by liquid chromatography mass spectrometry. Quantification was performed on the basis of linear calibration plots of peak area against concentration. Calibration lines were constructed based on five concentration levels of standard solutions.
2.3. Equipment and analytical conditions 2.3.1. HPLC–UV The HPLC system (Hewlett-Packard, CA, USA) consisted of an HP1100 quaternary pump, an HP1100 degassing device, a 20 L injection loop (Rheodyne, Cotati, CA, USA) an HP1100 UV-detector set at 215 nm. The HPLC was controlled by a personal computer running HP ChemStation software. Operating conditions were as follows: the chromatographic separation was performed on a TEKNOKROMA, Tr-010065 Mediterranea sea18 (15 cm × 0.4 cm, i.d. 3 m) and a TEKNOKROMA, Tr015605 TRACER EXTRASIL Octadecylsilane (ODS2) (25 cm × 0.4 cm, i.d. 5 m). The characteristics of the stationary phases regarding particle size (m); total carbon content (%); surface area (m2 /g); and average pore diameter (Å) are: 5 m; 12%; 200 m2 /g; and 80 Å for octadecylsilane (ODS2) column and 3 m; 17%; 450 m2 /g and 80 Å for Mediterranea sea18 , respectively. The mobile phase consisted of ultra-pure Milli-Q water containing 0.1% formic acid. The flow rate was set at 0.5 mL min−1 and the injection volume was 20 L. The analysis was performed at room temperature. 2.3.2. HPLC–MS: confirmation analysis A high performance liquid chromatograph 1100 Agilent coupled to a Microtof-Bruker spectrometer was used for organic acids identification. MS data were acquired in the negative ion electrospray ionization (ESI) mode. The operating conditions for ESI were: capillary voltage 4500 V and fragmentor voltage 70 V. The column and mobile phase were the same as in the HPLC–UV analysis.
3. Results and discussion Preliminary experiments were carried out in order to optimize the chromatographic conditions. Acidic mobile phases are common used in the analysis of carboxylic acids. Phosphoric acid is the typical eluent when a LC–UV system is employed, however this acid is not suitable for ESI since could damage the surface of the interface [17,19]. More volatile acids are required. Formic acid was selected because of their appropriate properties. On the one hand is compatible with ESI and on the other hand help to maintain a low pH. Two different proportions were tested (0.1 and 0.5% formic acid), the best separation was obtained with 0.1%, this is in agreement with a previous study conducted by Gamoh et al. [17]. Another essential step in the development of a chromatographic method is the selection of the stationary phase. In this study a conventional Tracer Extrasil Octadecylsilane (ODS2) and a novel Mediterranea sea18 were tested. The novel chromatographic support is based on perfectly spherical particles of extremely pure silica; with a very low metal content, additionally this packing is prepared to work with aqueous mobile phases in a wide pH range [20]. These characteristics make of this column an excellent alternative to the conventional octadecylsilane (ODS2) for the analysis of the organic acids. Both columns were compared concerning the sensitivity and system suitability parameters such as the number of the theoretical plates and the width of the peaks.
Fig. 1. Chromatograms of a standard solution performed on a Mediterranea sea18 (A) and on an octadecylsilane (ODS2) (B) columns using Milli-Q water containing 0.1% formic acid as mobile phase and at a flow rate of 0.5 mL min−1 . Peaks: (1) tartaric; (2) malic; (3) acetic; (4) lactic; (5) citric; (6) succinic.
A.R.-B. de Quirós et al. / Talanta 78 (2009) 643–646
645
Fig. 2. ESI-MS spectra: tartaric, malic, citric, succinic.
As can be seen from the data of Table 1 the use of the Mediterranea sea18 column lead to better performance and sensitivity as compared to the conventional octadecylsilane (ODS2) column, therefore this stationary phase was used for further assays. Chromatograms performed on a Mediterranea sea18 and on an octadecylsilane (ODS2) columns are presented in Fig. 1. The proposed analytical method was validated with regard to linearity, limits of detection and repeatabilities within day and between days. The linearity of the method was tested by using a series of organic acids standard solutions of known concentration. The calibration curves were constructed using five concentration levels and they were fitted to a linear equation. Each point of the calibration curve is the average of three peak–area measurements. Table 2 shows the linear equation, the range of linearity and the determination coefficients of the carboxylic acids studied. The method showed good linearity, determination coefficients were in all cases greater than 0.995. The detection limits (defined as a signal three times the high of the noise level) determined in accordance with the Analytical Chemical Subcommittee guidelines [21] are presented in Table 1. Lower limits of detection were obtained when using Mediterranea sea18 column.
The intra- and inter-day repeatabilities were determined by analyzing six replicates of the standards at three different concentration levels, expressed as the percentage of R.S.D. (%R.S.D. (n = 6)), in the same day and in different days, respectively. All analytes except acetic and malic acids presented a repeatability within day, expressed as the %R.S.D. (n = 6) lower than 1%; in what concerns to the repeatabilities between days the values obtained were lower than 3% except for acetic acid (Table 3). Organic acids were identified by comparing their retention times with the standards and confirmed by LC–MS. Characteristic masses (m/z) were 149, 133, 191, 117 and 89 for tartaric, malic, citric, sucTable 2 Equations of calibration curves. Analyte
Range of linearitya
Equation
R2
Tartaric Malic Lactic Acetic Citric Succinic
1–200 5–200 1–200 5–200 5–200 1–200
y = 3.100X(±0.005) − 0.7211(±0.518) y = 1.7506X(±0.07) − 7.0561(±6.97) y = 1.067X(±0.001) + 0.0490(±0.10) y = 6.6606X(±0.04) − 1.4363(±3.77) y = 3.1589X(±0.01) − 3.1478(±1.958) y = 1.467X(±0.0008) − 0.0390(±0.08)
0.999992 0.995 0.999997 0.99990 0.99994 0.9999991
a
mg/L.
646
A.R.-B. de Quirós et al. / Talanta 78 (2009) 643–646
Table 3 Repeatability within day and between days (%R.S.D. (n = 6)) at three concentrations levels.
analysis were performed directly without previous treatment. The amounts of organic acids found are presented in Table 4.
No.
Compound
1
Tartaric
2
Malic
3
Acetic
4
Lactic
5
Citric
6
Succinic
Concentration (ppm)
Intra-day %R.S.D. (n = 6)
Inter-day %R.S.D. (n = 6)
4. Conclusions
0.50 10 100 0.50 10 100 0.50 10 100 0.50 10 100 0.50 10 100 0.50 10 100
0.5 0.2 0.4 0.8 1 0.5 1 0.9 0.4 0.3 0.4 0.4 0.4 0.4 0.2 0.4 0.6 0.2
0.7 0.2 1.3 0.9 1 2 1 3 1.5 0.4 0.5 1.2 0.5 0.7 1.6 0.5 1.3 0.2
The new stationary phase could be an excellent alternative to the conventional octadecylsilane (ODS2) columns to analyse organic acids in different matrix. This novel packing showed an extraordinary sensitivity and a suitable performance. Acknowledgement A. Rodríguez-Bernaldo de Quirós is grateful to Xunta de Galicia for the award of a grant Parga Pondal. References
Table 4 Content of organic acids in samples analysed. Organic acid
Sample 1
Sample 2
Sample 3
Tartaric Malic Acetic Lactic Citric Succinic
191 147.6 68.2 57.1 149 36.7
193.7 157.7 67.5 57.6 155.5 37.9
198 147.5 69.8 57.7 147.8 36.9
cinic and lactic respectively. They correspond to the deprotonated molecular ion [M−H]− . Acetic acid could not be identified with the LC–MS system available because it is not suitable to analyse low weight molecules. The ESI-MS spectra corresponding to the tartaric, malic, citric and succinic acid are illustrated in Fig. 2. Once the chromatographic conditions were established and just to test the new column with real samples the proposed method was applied to the determination of organic acids in three different commercial available white wines from the same origin; the
[1] L.M.L. Nollet, Food Analysis by HPLC, Marcel Dekker, Inc., Basel, 2000. [2] C.I. Rodrigues, L. Marta, R. Maia, M. Miranda, M. Ribeirinho, C. Máguas, J. Food Compos. Anal. 20 (2007) 440. [3] G. Shui, L.P. Leong, J. Chromatogr. A 977 (2002) 89. [4] A. de Villiers, F. Lynen, A. Crouch, P. Sandra, Chromatographia 59 (2004) 403. [5] A. Kotani, Y. Miyaguchi, E. Tomita, K. Takamura, F. Kusu, J. Agric. Food Chem. 52 (2004) 52. [6] F. Silva, V. Ferraz, Food Chem. 88 (2004) 609. [7] M.Y. Ding, H. Koizumi, Y. Suzuki, Anal. Sci. 11 (1995) 239. [8] M. Castellari, A. Versari, U. Spinabelli, S. Galassi, A. Amati, J. Liq. Chrom. Rel. Technol. 23 (2000) 2047. [9] A. Edelmann, J. Diewok, J. Rodriguez Baena, B. Lendl, Anal. Bioanal. Chem. 376 (2003) 92. [10] M.I.H. Helaleha, K. Tanakaa, H. Taodaa, W. Hub, K. Hasebeb, P.R. Haddad, J. Chromatogr. A 956 (2002) 201. [11] Y. Soyer, N. Koca, F. Karadeniz, J. Food Compos. Anal. 16 (2003) 629. [12] M.J. Nozal, J.L. Bernal, J.C. Diego, L.A. Gómez, M. Higes, J. Liq. Chrom. Rel. Technol. 26 (2003) 1231. [13] N. Dinkci, A.S. Akalin, S. Gönc, G. Ünal, Chromatographia 66 (2007) S45. [14] T. Pérez-Ruiz, C. Martínez-Lozano, V. Tomás, J. Martín, J. Chromatogr. A 1026 (2004) 57. [15] A. Santalad, P. Teerapornchaisit, R. Burakham, S. Srijaranai, LWT 40 (2007 1741). [16] S.K. Johnson, L.L. Houk, J. Feng, D.C. Johnson, R.S. Houk, Anal. Chim. Acta 341 (1997) 205. [17] K. Gamoh1, H. Saitoh, H. Wada, Rapid Commun. Mass Spectrom. 17 (2003) 685. [18] C.W. Klampfl, Electrophoresis 28 (2007) 3362. [19] Z. Chen, X. Jin, Q. Wang, Y. Lin, L. Gan, C. Tang, J. Sep. Sci. 30 (2007) 2440. [20] Teknokroma Home Page http://www.teknokroma.es/mediterranea/. [21] American Chemical Society (ACS), Subcommittee of Environmental Analytical Chemistry, Anal. Chem. 52 (1980) 2242.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Short communication
HPLC analysis of organic acids using a novel stationary phase A. Rodríguez-Bernaldo de Quirós ∗ , M.A. Lage-Yusty, J. López-Hernández Analytical Chemistry, Nutrition and Bromatology Department, Pharmacy Faculty, Campus Sur s/n, University of Santiago de Compostela, 15782 Santiago de Compostela (La Coru˜ na), Spain
a r t i c l e
i n f o
Article history: Received 1 September 2008 Received in revised form 3 November 2008 Accepted 14 November 2008 Available online 21 November 2008 Keywords: Organic acids Stationary phase Liquid chromatography Mass spectrometry
a b s t r a c t In the present work, a high performance liquid chromatographic method with UV detection for the separation of six organic acids including, tartaric, malic, acetic, lactic, citric and succinic is described. The separation was performed on a novel stationary phase TEKNOKROMA, Tr-010065 Mediterranea sea18 (15 cm × 0.4 cm, i.d. 3 m) and using water with a 0.1% (v/v) of formic acid as mobile phase. The advantages of this packing over a conventional octadecylsilane (ODS2) column are reported. The method was validated with respect to linearity, limits of detection and repeatabilities within day and between days and satisfactory results were obtained. The proposed method was applied for the determination of these compounds in commercially available white wines. The samples were injected directly without previous treatment. LC–MS was used as a confirmatory technique. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The analysis of organic acids in different food items such as fruit juices, vegetables, dairy products, coffee and wine is of great interest for food industry since these compounds are responsible of sensory properties and may also influence their stability [1–5]. Regarding the analytical techniques, simple rapid and reliable methods are required for control purposes. Gas chromatography with flame ionization detector has been used to determine carboxylic acids in citric fruits [6] however this procedure presents a disadvantage, a derivatization step is necessary for non-volatile acids. Liquid chromatography with different separation mechanism including ion-exchange [4,7–9], ion-exclusion [7,10–12] and reversed phase [3,7,13,14] has been widely employed in different matrix. Nowadays capillary zone electrophoresis (CZE) with photodiode array detection appears as a promising analytical tool to determine these compounds in short time [15]. In complex samples mass spectrometry coupled to capillary electrophoresis or liquid chromatography constitutes a powerful technique due to its high sensitivity and selectivity [16–19]. In chromatography, the selection of the stationary phase is essential in order to achieve a suitable separation. In the present work a new reversed packing based on perfectly spherical par-
∗ Corresponding author. Tel.: +34 981 598450; fax: +34 981 594912. E-mail address: [email protected] (A.R.-B. de Quirós). 0039-9140/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2008.11.013
ticles of ultra pure silica and suitable for a wide pH range and aqueous mobile phases was used to analyse six organic acids including, tartaric, malic, lactic, acetic, citric and succinic. A comparison with a conventional octadecylsilane (ODS2) column in what concerns to system suitability parameters such as number of theoretical plates, and peaks width, and sensitivity is reported. The proposed method was validated in terms of linearity, limits of detection and repeatabilities within day and between days. In the second part of the work the method was applied to analyse white wine samples. LC–MS was used as a confirmatory technique. 2. Experimental 2.1. Chemicals and standard solutions All chemicals were of analytical grade. Formic acid (98–100%) was from Riedel-de Haën (Seelze, Germany) and meta-phosphoric acid was purchased from Sigma (St. Louis, MO, USA). Ultrapure water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA). Organic acid standards were obtained as follows: citric acid (≥99.5%) and dl-lactic acid (∼90%) were from Fluka BioChemika (Steinheim, Germany), malic acid (95–100%), succinic acid (99%) and dl-tartaric acid (99%) were from Sigma–Aldrich (St. Louis, MO, USA) and acetic acid (99–100%) was from Riedel-de Haën (Seelze, Germany). Stock standard solutions were dissolved in mobile phase, Milli-Q water (0.1% formic acid), and stored at 4 ◦ C in the dark.
644
A.R.-B. de Quirós et al. / Talanta 78 (2009) 643–646
Table 1 System suitability parameters and limits of detection of both stationary phases. Compound
Teknokroma ODS2 5 m column a
Width Tartaric Malic Lactic Acetic Citric Succinic a b c
0.20 0.24 0.38 0.21 0.37 0.41
± ± ± ± ± ±
Mediterranea sea18 3 m column b
0.04 0.13 0.09 0.01 0.02 0.01
c
Plates
LoD
Widtha
2281.7 2738.7 1324 4738.8 2335.1 2644.6
0.50 0.70 1.00 1.00 0.70 1.00
0.21 0.15 0.16 0.16 0.23 0.25
± ± ± ± ± ±
0.009 0.005 0.005 0.007 0.007 0.004
Platesb
LoDc
1679.11 5224.7 6500.2 7180.1 10729.9 10300.3
0.10 0.10 0.10 0.10 0.50 0.30
Peak-width (mean of 10 injections). Plates (mean of 10 injections). mg/L.
2.2. Sampling
2.4. Identification and quantification
Commercial available white wines from Galicia (NW Spain) and from the same vintage (2006) were analysed. The samples were filtered through a 50 m PTFE membrane filter (Advantec MFS, INC, CA, USA) and directly injected into the chromatograph without previous treatment. The analyses were performed in triplicate.
Identification of organic acids was made by comparison of their retention times with those of pure standards solutions. Moreover, citric acid, lactic acid, malic acid, succinic acid and tartaric acid were also confirmed by liquid chromatography mass spectrometry. Quantification was performed on the basis of linear calibration plots of peak area against concentration. Calibration lines were constructed based on five concentration levels of standard solutions.
2.3. Equipment and analytical conditions 2.3.1. HPLC–UV The HPLC system (Hewlett-Packard, CA, USA) consisted of an HP1100 quaternary pump, an HP1100 degassing device, a 20 L injection loop (Rheodyne, Cotati, CA, USA) an HP1100 UV-detector set at 215 nm. The HPLC was controlled by a personal computer running HP ChemStation software. Operating conditions were as follows: the chromatographic separation was performed on a TEKNOKROMA, Tr-010065 Mediterranea sea18 (15 cm × 0.4 cm, i.d. 3 m) and a TEKNOKROMA, Tr015605 TRACER EXTRASIL Octadecylsilane (ODS2) (25 cm × 0.4 cm, i.d. 5 m). The characteristics of the stationary phases regarding particle size (m); total carbon content (%); surface area (m2 /g); and average pore diameter (Å) are: 5 m; 12%; 200 m2 /g; and 80 Å for octadecylsilane (ODS2) column and 3 m; 17%; 450 m2 /g and 80 Å for Mediterranea sea18 , respectively. The mobile phase consisted of ultra-pure Milli-Q water containing 0.1% formic acid. The flow rate was set at 0.5 mL min−1 and the injection volume was 20 L. The analysis was performed at room temperature. 2.3.2. HPLC–MS: confirmation analysis A high performance liquid chromatograph 1100 Agilent coupled to a Microtof-Bruker spectrometer was used for organic acids identification. MS data were acquired in the negative ion electrospray ionization (ESI) mode. The operating conditions for ESI were: capillary voltage 4500 V and fragmentor voltage 70 V. The column and mobile phase were the same as in the HPLC–UV analysis.
3. Results and discussion Preliminary experiments were carried out in order to optimize the chromatographic conditions. Acidic mobile phases are common used in the analysis of carboxylic acids. Phosphoric acid is the typical eluent when a LC–UV system is employed, however this acid is not suitable for ESI since could damage the surface of the interface [17,19]. More volatile acids are required. Formic acid was selected because of their appropriate properties. On the one hand is compatible with ESI and on the other hand help to maintain a low pH. Two different proportions were tested (0.1 and 0.5% formic acid), the best separation was obtained with 0.1%, this is in agreement with a previous study conducted by Gamoh et al. [17]. Another essential step in the development of a chromatographic method is the selection of the stationary phase. In this study a conventional Tracer Extrasil Octadecylsilane (ODS2) and a novel Mediterranea sea18 were tested. The novel chromatographic support is based on perfectly spherical particles of extremely pure silica; with a very low metal content, additionally this packing is prepared to work with aqueous mobile phases in a wide pH range [20]. These characteristics make of this column an excellent alternative to the conventional octadecylsilane (ODS2) for the analysis of the organic acids. Both columns were compared concerning the sensitivity and system suitability parameters such as the number of the theoretical plates and the width of the peaks.
Fig. 1. Chromatograms of a standard solution performed on a Mediterranea sea18 (A) and on an octadecylsilane (ODS2) (B) columns using Milli-Q water containing 0.1% formic acid as mobile phase and at a flow rate of 0.5 mL min−1 . Peaks: (1) tartaric; (2) malic; (3) acetic; (4) lactic; (5) citric; (6) succinic.
A.R.-B. de Quirós et al. / Talanta 78 (2009) 643–646
645
Fig. 2. ESI-MS spectra: tartaric, malic, citric, succinic.
As can be seen from the data of Table 1 the use of the Mediterranea sea18 column lead to better performance and sensitivity as compared to the conventional octadecylsilane (ODS2) column, therefore this stationary phase was used for further assays. Chromatograms performed on a Mediterranea sea18 and on an octadecylsilane (ODS2) columns are presented in Fig. 1. The proposed analytical method was validated with regard to linearity, limits of detection and repeatabilities within day and between days. The linearity of the method was tested by using a series of organic acids standard solutions of known concentration. The calibration curves were constructed using five concentration levels and they were fitted to a linear equation. Each point of the calibration curve is the average of three peak–area measurements. Table 2 shows the linear equation, the range of linearity and the determination coefficients of the carboxylic acids studied. The method showed good linearity, determination coefficients were in all cases greater than 0.995. The detection limits (defined as a signal three times the high of the noise level) determined in accordance with the Analytical Chemical Subcommittee guidelines [21] are presented in Table 1. Lower limits of detection were obtained when using Mediterranea sea18 column.
The intra- and inter-day repeatabilities were determined by analyzing six replicates of the standards at three different concentration levels, expressed as the percentage of R.S.D. (%R.S.D. (n = 6)), in the same day and in different days, respectively. All analytes except acetic and malic acids presented a repeatability within day, expressed as the %R.S.D. (n = 6) lower than 1%; in what concerns to the repeatabilities between days the values obtained were lower than 3% except for acetic acid (Table 3). Organic acids were identified by comparing their retention times with the standards and confirmed by LC–MS. Characteristic masses (m/z) were 149, 133, 191, 117 and 89 for tartaric, malic, citric, sucTable 2 Equations of calibration curves. Analyte
Range of linearitya
Equation
R2
Tartaric Malic Lactic Acetic Citric Succinic
1–200 5–200 1–200 5–200 5–200 1–200
y = 3.100X(±0.005) − 0.7211(±0.518) y = 1.7506X(±0.07) − 7.0561(±6.97) y = 1.067X(±0.001) + 0.0490(±0.10) y = 6.6606X(±0.04) − 1.4363(±3.77) y = 3.1589X(±0.01) − 3.1478(±1.958) y = 1.467X(±0.0008) − 0.0390(±0.08)
0.999992 0.995 0.999997 0.99990 0.99994 0.9999991
a
mg/L.
646
A.R.-B. de Quirós et al. / Talanta 78 (2009) 643–646
Table 3 Repeatability within day and between days (%R.S.D. (n = 6)) at three concentrations levels.
analysis were performed directly without previous treatment. The amounts of organic acids found are presented in Table 4.
No.
Compound
1
Tartaric
2
Malic
3
Acetic
4
Lactic
5
Citric
6
Succinic
Concentration (ppm)
Intra-day %R.S.D. (n = 6)
Inter-day %R.S.D. (n = 6)
4. Conclusions
0.50 10 100 0.50 10 100 0.50 10 100 0.50 10 100 0.50 10 100 0.50 10 100
0.5 0.2 0.4 0.8 1 0.5 1 0.9 0.4 0.3 0.4 0.4 0.4 0.4 0.2 0.4 0.6 0.2
0.7 0.2 1.3 0.9 1 2 1 3 1.5 0.4 0.5 1.2 0.5 0.7 1.6 0.5 1.3 0.2
The new stationary phase could be an excellent alternative to the conventional octadecylsilane (ODS2) columns to analyse organic acids in different matrix. This novel packing showed an extraordinary sensitivity and a suitable performance. Acknowledgement A. Rodríguez-Bernaldo de Quirós is grateful to Xunta de Galicia for the award of a grant Parga Pondal. References
Table 4 Content of organic acids in samples analysed. Organic acid
Sample 1
Sample 2
Sample 3
Tartaric Malic Acetic Lactic Citric Succinic
191 147.6 68.2 57.1 149 36.7
193.7 157.7 67.5 57.6 155.5 37.9
198 147.5 69.8 57.7 147.8 36.9
cinic and lactic respectively. They correspond to the deprotonated molecular ion [M−H]− . Acetic acid could not be identified with the LC–MS system available because it is not suitable to analyse low weight molecules. The ESI-MS spectra corresponding to the tartaric, malic, citric and succinic acid are illustrated in Fig. 2. Once the chromatographic conditions were established and just to test the new column with real samples the proposed method was applied to the determination of organic acids in three different commercial available white wines from the same origin; the
[1] L.M.L. Nollet, Food Analysis by HPLC, Marcel Dekker, Inc., Basel, 2000. [2] C.I. Rodrigues, L. Marta, R. Maia, M. Miranda, M. Ribeirinho, C. Máguas, J. Food Compos. Anal. 20 (2007) 440. [3] G. Shui, L.P. Leong, J. Chromatogr. A 977 (2002) 89. [4] A. de Villiers, F. Lynen, A. Crouch, P. Sandra, Chromatographia 59 (2004) 403. [5] A. Kotani, Y. Miyaguchi, E. Tomita, K. Takamura, F. Kusu, J. Agric. Food Chem. 52 (2004) 52. [6] F. Silva, V. Ferraz, Food Chem. 88 (2004) 609. [7] M.Y. Ding, H. Koizumi, Y. Suzuki, Anal. Sci. 11 (1995) 239. [8] M. Castellari, A. Versari, U. Spinabelli, S. Galassi, A. Amati, J. Liq. Chrom. Rel. Technol. 23 (2000) 2047. [9] A. Edelmann, J. Diewok, J. Rodriguez Baena, B. Lendl, Anal. Bioanal. Chem. 376 (2003) 92. [10] M.I.H. Helaleha, K. Tanakaa, H. Taodaa, W. Hub, K. Hasebeb, P.R. Haddad, J. Chromatogr. A 956 (2002) 201. [11] Y. Soyer, N. Koca, F. Karadeniz, J. Food Compos. Anal. 16 (2003) 629. [12] M.J. Nozal, J.L. Bernal, J.C. Diego, L.A. Gómez, M. Higes, J. Liq. Chrom. Rel. Technol. 26 (2003) 1231. [13] N. Dinkci, A.S. Akalin, S. Gönc, G. Ünal, Chromatographia 66 (2007) S45. [14] T. Pérez-Ruiz, C. Martínez-Lozano, V. Tomás, J. Martín, J. Chromatogr. A 1026 (2004) 57. [15] A. Santalad, P. Teerapornchaisit, R. Burakham, S. Srijaranai, LWT 40 (2007 1741). [16] S.K. Johnson, L.L. Houk, J. Feng, D.C. Johnson, R.S. Houk, Anal. Chim. Acta 341 (1997) 205. [17] K. Gamoh1, H. Saitoh, H. Wada, Rapid Commun. Mass Spectrom. 17 (2003) 685. [18] C.W. Klampfl, Electrophoresis 28 (2007) 3362. [19] Z. Chen, X. Jin, Q. Wang, Y. Lin, L. Gan, C. Tang, J. Sep. Sci. 30 (2007) 2440. [20] Teknokroma Home Page http://www.teknokroma.es/mediterranea/. [21] American Chemical Society (ACS), Subcommittee of Environmental Analytical Chemistry, Anal. Chem. 52 (1980) 2242.