1. Introduction
There is a growing interest in separation science to achieve dramatic increases in resolution, speed and sensitivity in liquid chromatography. Significant advances in technology were made for ultra-fast separation with high efficiency through an Ultra-Performance Liquid Chromatography (UPLC Technology) by Waters [1].
UPLC has overcame the negative aspect of packed columns used in high-perfor-mance liquid chromatography (HPLC) by precisely delivering mobile phase at pressures up to 15,000 psi, thus enabling columns packed with smaller particles (sub-2 m) to provide higher level of chromatographic performance [2]. Besides, because of the separation methods are significantly faster, UPLC technology reduces the use of eluents and wastes compared with conventional HPLC [3].
Furthermore, the UPLC system facilitates a low limit of detection since the signal--to-noise ratio is improved and the injection volume can be considerably reduced without losing the sensitivity [4].
In order to compare the performance of HPLC and UPLC, analyses for the determination of melatonin ( -acetyl-3-(2-aminoethyl)-5-methoxyindole; Fig. 1)
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Comparison of HPLC and UPLC methods for
using both methods were studied. Research into melatonin continued with many results revealing the utility of this compound. Melatonin diminishes neuro-degenerative diseases, such as Parkinson and Alzheimer [5]; it also acts as an anticancer agent [6]. Recently, research articles have appeared discussing methods of analysis of melatonin in different matrices; these methods include HPLC-EC [7, 8], HPLC-UV [9–11], HPLC-MS [12], ELISA [13–15], and GC-MS [16].
Although melatonin determination in different matrices has been reported, limited studies performed on separation of melatonin by UPLC, particularly in conjunction with fluorescence detection (FD). The aim of this study was to develop a new separation method by UPLC-FD and to compare with conventional HPLC for analysing melatonin in rice grain extracts.
HPLC grade methanol, acetonitrile, and acetic acid were purchased from Merck..
Melatonin standard was obtained from Sigma-Aldrich. Water was purified with a Milli-Q purification system (Millipore, USA).
Melatonin extraction was performed on a basis of microwave-assisted extraction (MAE) technique in a Milestone Ethos 1600 (Sorisole, Italy) equipped with the vessels which are made of tetrafluoromethoxyl and lined with Teflon liners. The extraction of melatonin from rice grains was conducted according to the esta-blished procedure [17].
HPLC analyses were carried out on an Alliance HPLC 2695 system with a fluores-cence detector (Waters 474 fluoresfluores-cence detector), controlled by an Empower Pro 2002 data station (Waters, Milford, MA, USA). Separations were performed in 2. Experimental
2.1 Reagents and chemicals
2.2 Extraction of melatonin from rice grains
2.3 HPLC conditions
Fig. .1Chemical structure of melatonin.
a reverse phase RP 18 Lichrospher Column (LiChroCART 250×4 mm (5 m) from Merck) at 25 °C. The mobile phase was a binary solvent system consisting of phase A (2% acetic acid and 5% methanol in water) and phase B (2% acetic acid and 88%
methanol in water) with a flow rate of 0.5 mL min . The 25 min programmed gradient was as follows (%B): 0−5 min, 0−35%; 5−12 min, 35−40%; 12−15 min, 40%; 20−25 min, 45−50%. After each analysis, the column was washed with 100% B for 5 min and equilibrated with 0% B for 5 min. The established con-ditions for the fluorescence detector were as follows: excitation wavelength, 290 nm; emission wavelength, 330 nm; gain, 1000; attenuation, 16; injection volume, 10 L.
UPLC analyses were carried out on an ACQUITY UPLC® H-Class system coupled to an ACQUITY UPLC Fluorescence Detector (FD) and controlled by Empower™ 3 Chromatography Data Software (Waters Corporation). The excitation wavelength was set at 290 nm and the emission wavelength was set at 330 nm for the 2D scan.
The FD sensitivity for the 2D scan was set at PMT gain 1, the data rate at 40 pts s and the time constant at 0.1 s. Separations were performed at a temperature of 47 °C on a reverse phase RP 18 Acquity UPLC® BEH Column (Acquity UPLC® BEH 100 2.1 (1.7 m) from Waters Corporation).
The mobile phase was a binary solvent system consisting of phase A (water with 2% acetic acid) and phase B (acetonitrile with 2% acetic acid). The flow rate was 0.7 mL min . The 4.0 minute gradient was as follows (%B): 0−1 min, 0%;
1−1.1 min, 0−10%; 1.1−2 min, 10%; 2−3 min, 10−20%; 3−3.5 min, 20−60%;
3.5−4 min, 60−100%. The column was subsequently washed with 100% B for 3 min and equilibrated with 0% B for 3 min. The injection volume was set at 3.0 L.
The analytical procedure of the chromatographic method for melatonin deter-mination was carried out according to the recommendations of ICH Guideline Q2 (R1) [18] and suggestions in ISO 17025 [19]. Linearity, range, precision, detection and quantification limits of the method were established. The stability test inside the auto-sampler up to 4h for melatonin in standard solution (750 g L ) was investigated. The peak signal was determined every 30 minutes and compared with those obtained from freshly prepared melatonin solution.
In this study, UPLC method was developed and compared to the established HPLC method for the determination of melatonin in rice grain extracts. Different injec-tion volumes, flow-rates and column temperatures were evaluated during the
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2.4 UPLC analysis
2.5 Performance of the method
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3. Results and discussion
development of the UPLC method. To reach equivalent system performance compared with conventional HPLC, UPLC instrument manufacturer recommends using a low injection volume and 3.0 L was preferred for the analysis. The retention time of melatonin decreased with the increase of column temperature as well as the peak resolution. Hence, the column temperature of 47 °C was chosen based on the maximum column temperature previously developed by the research group. At this column temperature, a rapid separation was achieved in less than 4 min using the flow rate of 0.7 mL min . Fig. 2 shows the identification of melatonin in a standard solution (A) and rice grain extract (B) at the retention time of 21.2 and 3.4 min by HPLC and UPLC respectively. Apparently, the analysis run time reduced by two-thirds applying higher-pressure technology, which saved the eluents consumption up to 60%.
The validation properties for both chromatographic methods are listed in Table 1. The linearity of the calibration curves was validated by the high value of coefficient of determination of the regression analysis ( >0.99) for both chro-matographic methods. The standard deviation and slope obtained from the regression analysis were used to calculate the limit of detection ( ) and limit of quantification (LOQ). Since both limits were lower in UPLC than those in HPLC it is considered that the UPLC method is able to perform analysis in lower concentration of melatonin.
The precisions of the methods were evaluated by assessing repeatability (intra--day) and intermediate precision (extra(intra--day). The precisions, expressed as coefficient of variantion (CV), of retention time were prominently better for UPLC
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Fig. 2.Chromatograms of melatonin obtained by HPLC (left) and UPLC (right) analysis in standard solution (A) and in rice extract (B).
analysis. Conversely, the peak-height repeatability were similar for both methods while the intermediate precision was slightly better for UPLC method. Hence, the analysis using UPLC is considerably more precise than using conventional HPLC method.
The stability of melatonin in standard solution of 50% methanol/water (v/v) was analysed by UPLC-FD during 4 h inside the auto-sampler. CVs of the peak area were roughly 15% applying regular temperature of auto-sampler (15 °C). There fore, further studies evaluating lower temperature (4 C), the use of antioxidants (50 ppm SO and 5 ppm gallic acid) and direct injection of the solution from 20 C storage location were performed. Fig. 3 shows the use of gallic acid could help to maintain the stability of melatonin during the time in auto-sampler waiting for the injection.
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Parameter HPLC UPLC
Linear range concentration g L 0.5–15 0.5–20 C
g L 1.15 0.73
g L 3.84 2.19
Precisions-retention time
Repeatability ( = 9), CV % 0.99 0.28
Intermediate precisions ( = 3 3), CV % 1.23 0.12 Precisions-signal of height
Repeatability ( = 9), CV % 0.93 0.97
Intermediate precisions = 3 3), CV % 2.05 1.40
[ ]
[ ]
[ ]
[ ]
× [ ]
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( × [ ]
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oefficient of determinationR2 0.996 0.999
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LOD LOQ
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n Table 1
Method validation parameters for determination of melatonin in rice grain extracts.
Fig. .3Stability study of melatonin solution in auto-sampler.
4. Conclusions
Although several methods have been developed for determination of melatonin, both HPLC and UPLC proposed in this study can be recommended as rapid, precise and reliable methods for the determination of melatonin in rice grain extracts.
However, UPLC can offer significant enhancements in speed and precisions compared with conventional HPLC. This technique is also more eco-friendly due to the lower consumption of eluent.
Acknowledgments
References
W.S. is grateful to the CIMB Foundation for a Ph.D. studentship through the CIMB Regional Scholar-ships 2012.
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1. Introduction
Peptides are one of the most important groups of compounds, which are nece-ssary for the normal growing and functioning of every organism. The structure of the peptide is formed by a linear sequence of amino acids linked by peptide bonds.
The number, structure and sequences of amino acids determine the biological activity of the polypeptide molecule [1].
Specific properties of different types of peptides cause widespread interest and applications in many fields of science, e.g. endocrinology [2], cosmetology [3], pharmaceutical and medical industry [4, 5]. Peptides due to their unique struc-tural and biological properties exhibit also analytical application in liquid chromatography. The initiators of the chemical binding of the peptides on the surface of silica gel were Grushka and Scott [6, 7]. Commonly, peptide stationary phases are used in the analysis of both amino acids and dipeptides, as well as their isomers and derivatives. Amphoteric structure of amino acids makes it possible to use this type of materials as the zwitterionic stationary phases [8]. Peptide adsorbents significantly increase the collection of materials with a high ortho-gonality in two-dimensional mode RP/RP LC [9].
The application of peptide stationary phases currently involves a complex procedure for the synthesis of such materials. An interesting alternative synthesis