ˇ ecˇinsk, Bartk, Strnad, Prinsen Rolcˇk, R
1370 Jakub Rolcˇk1 ˇ ecˇinsk2 Jana R Petr Bartk2 Miroslav Strnad1 Els Prinsen3 1
Laboratory of Growth Regulators, Palack y University & Institute of Experimental Botany ASCR, Olomouc, Czech Republic 2 Department of Analytical Chemistry, Palack y University, Olomouc, Czech Republic 3 Department of Biology, University Antwerp, Antwerp, Belgium
Purification of 3-indolylacetic acid by solid phase extraction This paper deals with the use of solid-phase extraction (SPE) for the extraction and purification of 3-indolylacetic acid (IAA), the main and most important representative of the plant growth regulators auxins. The procedure is based on the use of C18-SPE columns in two steps. In the first one, raw extract in methanol:water (4:1) is applied on the column in order to remove neutral ballast. In the second step the eluate is diluted with water to a final methanol concentration of 20% (to decrease the elution strength of the solvent) and acidified with formic acid to a final concentration of 1% (to convert the IAA from the anionic to the neutral form). Neutral IAA is then retained on the second SPE column, eluted by (acidified) methanol, and subjected to final analysis. Scintillation counting of tritium labeled IAA standard was used for the investigation and optimization of the SPE procedure. Gas chromatography with mass spectrometric detection was suggested as a convenient method for routine determination of IAA after its methylation with diazomethane. Overall recovery of the procedure was estimated as 89 – 94% and a physiological level of IAA equal to 0.48 nmol/g (84 ng/g) fresh weight was found using an optimized SPE-GC-MS method. Key Words: Auxin; Indolylacetic acid; GC; Solid-phase extraction; Received: April 26, 2005; revised: May 26, 2005; accepted: May 27, 2005 DOI 10.1002/jssc.200500189
1 Introduction 3-Indolylacetic acid (IAA) is a naturally occurring substance from a group of plant hormones denoted as auxins. It is involved in the regulation of many processes of plant growth and development, including apical dominance, root growth, branching, and cell division and elongation. Although IAA has been known for more than 70 years [1, 2] and has been the subject of intense research, many questions concerning IAA biosynthesis, transport, and distribution in plants remain unanswered [3, 4]. The analytical chemistry of IAA and its continuous improvement therefore still retain their importance. There are several techniques for estimating IAA in plant material. HPLC coupled to either fluorescence [5] or coulometric [6] detection has been described. Another method employs immunoassay [7]. The use of gas chromatography was also reported [8]. This requires a prior derivatization of IAA, e.g. pentafluorobenzylation [9], methylation [10, 11], trimethylsilylation [12], or other procedures [13] leading to an enhanced volatility and, with respect to mass detection, higher molecular mass of the Correspondence: Dr. Jakub Rolcˇk, Laboratory of Growth Regulators, Palacky University & Institute of Experimental Botany ˇ lechtitelu˚ 11, 783 71 Olomouc, Czech Republic. ASCR, S Phone: +420 585 634 864. Fax: +420 585 634 870. E-mail:
[email protected]. Abbreviation: IAA, 3-Indolylacetic acid.
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analyte. The sweeping progress in analytical instrumentation in recent years has also permitted the quantification of IAA and its metabolites by HPLC coupled to tandem mass spectrometry, in materials of both bacterial and plant origin [14, 15]. Here, amounts as low as 50 mg are sufficient for the analysis. Aside from the instrumentation improvement, each final analysis has to be preceded by suitable extraction and purification procedure, typically comprising solid-phase extraction (SPE). Irrespective of the solution used for extraction (which may be phosphate buffer of pH 6.5 [16, 17], absolute methanol [18], 90% methanol [19], or 80% methanol [20]), such SPE is based on the retention of the protonated form of IAA on a non-polar C18 matrix. To achieve this, methanolic extracts are reduced to an aqueous phase by evaporation in vacuo and, similarly to phosphate buffer extracts, acidified to shift the acid-base balance of IAA in favour of its uncharged form. Subsequent elution of retained IAA is usually performed by application of either absolute [17, 19] or 80% methanol [18] or by application of 80% methanol containing 1% of acetic acid [15]. In the present work we concentrated on IAA separation by C18-based SPE so that it could be incorporated into an analytical protocol starting with extraction of IAA from plants with 80% methanol [21]. Further purification of the extract consisted in removing chlorophylls and other nonpolar ballast compounds by passing the sample through a
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Purification of 3-indolylacetic acid by solid phase extraction non-polar (C18) column followed by the studied SPE based on an interaction of the C18 matrix with protonated IAA. Final analysis was done by GC-MS of methylated SPE-purified analyte.
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porated under a nitrogen stream, reconstituted in 400 lL of methanol, centrifuged, and then analyzed by GC/MS.
2.3 Solid-phase extraction 2.1 Materials 3-Indolylacetic acid was obtained from Sigma (St. Louis, USA). 3-[5(n)-3H]Indolylacetic acid with a specific activity of 962 GBq/mmol was purchased from Amersham Biosciences (Little Chalfont, UK). The radiochemical purity of tritium-labeled IAA, checked by high performance liquid chromatography coupled to fluorescence and radioactive detection (data not shown), was about 96%. Ultima Gold scintillation cocktail was ordered from Perkin Elmer Life and Analytical Sciences (Boston, USA). HPLC-grade methanol and water were purchased from Acros Organics (Geel, Belgium). All the other solvents and chemicals were of reagent grade, provided either by Sigma (St. Louis, USA) or Acros Organics (Geel, Belgium). C18 SPE columns (BondElutm, 500 mg) were obtained from Varian (Harbor City, USA). Leaves of orchid containing less than 10 pmol of IAA per gram of fresh weight were used as a “zero” matrix. The level of IAA was assessed by a procedure described earlier [21]. Leaves of pelargonium of average surface area around 40 cm were used as a real sample.
2.2 Processing of plant material Plant material (0.5 g FW) was frozen in liquid nitrogen, ground with a pestle and mortar, and extracted for 1 hour with 4 mL of 80% (v/v) methanol containing 2.5 mM sodium diethyldithiocarbamate ( – 208C). The extract was centrifuged (30 000 g, 20 min, 48C) and passed through a C18 column preconditioned with absolute and 80% (v/v) methanol. The column was then rinsed with 1 mL of 80% (v/v) methanol and the eluates pooled.
To assess the highest applicable methanol concentration ensuring satisfactory IAA retention on the C18 matrix, a set of five 20-mL solutions, each containing 909 pmol of IAA, ca 850 Bq of 3H-IAA, 1% (v/v) of formic acid, and different concentrations of methanol (15, 20, 25, 30, and 40 per cent; v/v), was prepared. The solutions were applied on preconditioned C18 columns, which were subsequently rinsed with 5 mL of formic acid (1%) containing methanol at corresponding concentrations. Retained IAA was eluted with 1.5 mL of a mixture of methanol (70%) and formic acid (1%). The volumes of the eluates were adjusted to 10 mL with absolute methanol. 500-lL aliquots of each solution were taken for liquid scintillation counting. The experiment was performed in triplicate. In experiments aiming to assess an optimal methanol concentration in the eluting agent, six 5-mL portions of 80% (v/v) methanol (or 5 mL of orchid leaf extract described above) were diluted with 15 mL of 1.33% (v/v) formic acid and 909 pmol of unlabeled IAA and approximately 850 Bq of 3H-IAA were added. The solutions were applied to pretreated C18 columns, which were subsequently washed with 5 mL of 20% methanol containing 1% (v/v) formic acid. IAA was eluted from the respective columns with 1.5 mL of the following solutions: absolute methanol, methanol containing 1% (v/v) of formic acid, and a solution of 1% formic acid containing 90, 80, 70, and 60% (v/v) of methanol, respectively. The volumes of the respective eluates were made up to 10 mL with absolute methanol and 500-lL aliquots were taken for liquid scintillation counting. The experiment was performed in triplicate. A Packard Tri-Carb 1500 liquid scintillation analyzer (Packard Instrument Company, Downers Grove, USA) was used for liquid scintillation counting of samples mixed with 4 mL of scintillation cocktail.
The obtained orchid leaf extract served as a “zero” matrix in a study of SPE (see Section 2.3). The extract of pelargonium was diluted with 1.33% (v/v) formic acid to a volume of 20 mL and passed through a preconditioned C18 column. The column was washed with 5 mL of 20% methanol containing 1% (v/v) formic acid and retained IAA was eluted with 1.5 mL of absolute methanol, methanol acidified with formic acid (1%; v/v), or with an aqueous solution of 1% formic acid containing 90, 80, 70, and 60 per cent (v/v) of methanol, respectively. The eluate was dried under a nitrogen stream (378C) and treated with 500 lL of ethereal diazomethane freshly prepared according to [22]. After 20 min the mixture was evaJ. Sep. Sci. 2005, 28, 1370 – 1374
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2.4 Gas chromatography-mass spectrometry An HP 6890 Series GC system with 5973 N Mass Selective Detector and HP 7683 Series Injector (Agilent, Palo Alto, CA, USA) was used for analysis. An HP-5 MS column (30 m60.25 mm60.25 lm) was run through a temperature program: 508C for 2 min ramped at 10 K min – 1 up to 3008C, for 10 min with helium as carrier gas (99.998%, 0.9 mL min – 1, SIAD, Bergamo, Italy). The injection volume was 1 lL for all types of samples using the pulsed splitless mode (injection pulse: 20 psi, 0.4 min, injector temperature 2708C).
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Short Communication
2 Experimental
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3 Results and discussion 3.1 C18-based solid-phase extraction of 3-indolylacetic acid The sample preparation procedure, an integral part of which is the solid-phase extraction (SPE) described in the present paper, consists of IAA extraction from plant material with 80% methanol, removal of non-polar compounds by passing the extract through a non-polar sorbent, and subsequent SPE. Final analysis is then performed by GCMS. We used the C18 matrix for both the purification steps (removal of non-polar compounds and SPE-pre-concentration). However, there is a substantial difference in the way in which the non-polar character of this matrix is exploited. In the first purification step the non-polar compounds (pigments and lipids) extracted with 80% methanol from the plant sample together with the analyte are retained on C18 sorbent while ionized, and hence polar, IAA passes through the column. As a mater of fact, in relation to the analyte the procedure cannot be regarded as solid-phase extraction. The case of the second purification step is distinctly different, being based on IAA retention on C18 sorbent from acidified extract, freed of non-polar ballast compounds in the previous step. Such solid-phase extraction based on interaction of C18 matrix with uncharged form of IAA has already been described, as part of a purification protocol for quantification of IAA in plants [18]. However, execution of the method often differs in details. Thus the use of both phosphoric [17] and acetic acid [19] for acidification of plant extract prior to SPE was reported. Elution of retained IAA was accomplished by using either absolute [17, 19] or 80% methanol [18]. The use of 80% methanol containing 1% of acetic acid was also described [15]. Kowalczyk and Sandberg used the same solution to elute IAA from Isolute Env+ sorbent [23]. In all our experiments we preferred to use formic acid due to its volatility. Typically, the plant extract of IAA in 80% methanol is acidified only after it has been transformed into an aqueous phase by vacuum evaporation [18, 19, 23]. To eliminate this time-consuming step and replace it by a simple dilution procedure, we studied the influence of various methanol concentrations on IAA retention on the C18 matrix. We applied tritium-labeled IAA standard in solutions of various methanol concentrations (v/v; 15, 20, 25, 30, and 40%, respectively), each containing 1% (v/v) of formic acid, to C18 columns and eluted the retained analyte with a mixture of methanol (70%) and formic acid (1%), which ensures efficient elution (see below). Figure 1 summarizes the results based on liquid scintillation counting of respective eluates. It is clear that the methanol concentration in the applied solution has to be J. Sep. Sci. 2005, 28, 1370 – 1374
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Figure 1. Dependence of IAA retention on the concentration of methanol in IAA solution applied on C18 matrix. Tritiumlabeled IAA was applied in aqueous solutions containing formic acid (1%, v/v) and methanol at various concentrations (15, 20, 25, 30, and 40%, v/v, respectively). Elution was performed by applying 1.5 mL of 70% methanol containing 1% (v/v) of formic acid. Eluates were measured by liquid scintillation counting.
lowered to 20% (v/v) to achieve satisfactory distribution of protonated IAA favouring its retention on the C18 matrix. As we use 80% (v/v) methanol to extract IAA from plant material, one volume of the extract has to be diluted with three volumes of aqueous formic acid which is much simpler than the methanol evaporation commonly used to reach the same goal. We also believe that methanol present in an applied solution may have some positive effect on the distribution of non-polar ballast compounds, leading to their reduced extraction by non-polar sorbent. The most efficient way of eluting IAA retained on C18 matrix was another subject of our study. Tritium-labeled IAA standard was applied to a set of C18 columns and then eluted from each respective column with a 1.5-mL portion of one of the following solutions: absolute methanol, methanol containing 1% (v/v) of formic acid, and aqueous formic acid (1%) containing 90, 80, 70, and 60 per cent (v/v) of methanol, respectively. The results based on liquid scintillation counting of eluted radioactivity are shown in Figure 2. Recovery of IAA eluted with methanol acidified with formic acid (1%, v/v) was similar for methanol concentrations 100, 90, and 80%, respectively, varying around 94%. A considerably higher amount of IAA was eluted when an acidified solution of 70% methanol was used. Here we found approxi-
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because of its low eluting efficiency (92%) and because the higher content of water prolongs the requisite evaporation step. Noteworthy is a comparison of the recoveries achieved by eluting IAA with absolute methanol and with methanol acidified with formic acid (99:1; v/v), respectively. The recovery on use of acidified methanol was 94% while that obtained with absolute methanol amounted to only 86%. It is apparent that the maintenance of acidic conditions keeping IAA molecule uncharged has a significant impact on the distribution equilibrium of IAA between C18 sorbent and methanolic environment. This finding should be taken into account when the highest volatility of eluate, with respect to its subsequent evaporation, is required.
Figure 2. Elution efficiency of IAA retained on C18 column. Tritium-labeled IAA was applied in an aqueous solution of formic acid (1%, v/v) and methanol (20%, v/v). Elution was performed either with absolute methanol (MeOH) or with a solution of formic acid (1%) containing from 60 to 100% of methanol, respectively. Eluted radioactivity was assessed by liquid scintillation counting.
mately 102% recovery, which may be of considerable importance when low levels of IAA are to be quantified and the highest possible recoveries required. Besides, the result indicates that there is no need for repeated elution. On the other hand, an acidified solution containing 60% methanol seems to be of little use as an elution agent,
The same experiment performed with a mixture of IAA standard and an orchid leaf extract serving as a “zero” matrix gave almost identical result (data varying within experimental error were obtained). The fact corroborates our findings and points out their importance for practical utilization.
3.2 Determination of 3-indolylacetic acid A quantification procedure comprising SPE on a C18 sorbent was used to determine the IAA level in pelargonium leaves. GC-MS was selected as a convenient analytical tool for the purpose. Free IAA isolated by the SPE procedure was transformed to its methyl ester by reaction with diazomethane (see Experimental). The efficiency of the derivatization procedure was monitored by the direct comparison of the peak area of derivatized IAA with the peak area obtained by analysis of the standard solution of IAA methyl ester and was estimated to be 97 – 98% in the pre-
Figure 3. GC-MS chromatogram obtained from pelargonium, SIM (130 m/z). IAA concentration was found to amount to 0.47 nmol/g of fresh weight. Retention time of IAA methyl ester (IAAMe) is 18.4 min. J. Sep. Sci. 2005, 28, 1370 – 1374
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sence of “zero” sample matrix. Similarly, the recovery of the SPE procedure was estimated by the standard addition method. Comparison of the samples enriched with IAA before the SPE procedure with the peak area obtained by analysis of the standard solution of IAA methyl ester gives the recovery of the overall procedure as 89 – 94%. Finally the concentration of IAA in natural material was determined using both the standard addition method and the absolute calibration corrected by the recovery value. Concentrations of 0.47 nmol/g and 0.49 nmol/g of fresh weight were obtained by the respective calibration methods. The use of our findings in development of a more complex purification procedure combining other separation techniques (e.g. immunoaffinity extraction) is a matter of further research.
4 Concluding remarks C18-based solid-phase extraction of 3-indolylacetic acid was studied. The method consists in retention of uncharged protonated molecule of IAA on non-polar C18 matrix. Here we concentrated on the use of 1% (v/v) formic acid in aqueous methanol and studied the impact of various methanol concentrations on both retention and elution of IAA. To achieve satisfactory retention of IAA, the methanol content should not exceed 20% (v/v). For elution of retained IAA, methanolic solutions acidified with formic acid should be used to obtain either 94% recovery (for acidified absolute methanol) or recovery close to 100% (for 70% methanol). Where the highest recovery is required, because of a complex purification protocol and minimum analyte concentrations in the sample, the use of absolute methanol alone cannot be recommended as it leads to a recovery of about 86% only. We also report that the non-polar character of the C18 sorbent may well be exploited in a purification step preceding the described solid-phase extraction. In this instance purification is based on the polar character of the (presumably dissociated) molecule of IAA in 80% aqueous methanol, which passes through the column while non-polar ballast compounds are retained on the non-polar C18 matrix. We employed both the purification steps based on the dual use of C18 sorbent in IAA analysis in pelargonium, thus demonstrating the usefulness of this sorbent, which is often used in analytical laboratories. The IAA level found in pelargonium by GC-MS was about 0.48 nmol/g (84 ng/g) fresh weight.
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Acknowledgement The work was supported by the Ministry of Education, Youth, and Sports of the Czech Republic grants No.: 1K03021 and MSM 6198959216. We thank Olga Hustkov for excellent technical assistance. We are grateful to Dr. Karel Sigler and Dr. Radomra Vanˇkov for careful revision of the manuscript.
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