Computer-Assisted Optimization of a Gradient HPLC Method for the Separation of Flavonoids

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25 Computer-Assisted Optimization of a Gradient HPLC Method for the Separation of Flavonoids Tomasz Baczek, Grazyna Lewandowska, Roman Kaliszan, Department of Biopharmaceutics and Pharmacodynamics,

1 25 Computer-Assisted Optimization of a Gradient HPLC Method for the Separation of Flavonoids Tomasz Baczek, Grazyna Lewandowska, Roman Kaliszan, Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdansk, Poland, Miroslawa Krauze-Baranowska and Wojciech Cisowski, Department of Pharmacognosy, Medical University of Gdansk, Poland A high-performance liquid chromatography procedure based on the linear gradient elution technique was used to separate flavonoids present in methanolic extracts from Taxus baccata var. elegantissima and Metasequoia glyptostroboides. Optimization of chromatographic separations was obtained by means of a computer. Chromatographic separation parameters were computer optimized and these predicted conditions were then successfully applied to actual separations. Predictive errors were calculated and a satisfactory correlation between the predicted and experimental retention data was found. Computer-assisted optimization with the use of linear gradients is able to provide good chromatographic separations of flavonoids in an easy, inexpensive and reproducible manner. Introduction Flavonoids have recently received increased interest as food and beverage components and additives. The reason is their reported preventative activity in circulatory diseases and post-climacteric osteoporosis (1, 2). Hence, there is a need for a convenient, widely applicable and inexpensive method of their determination. Flavonoids are a class of compounds that are widespread in plants all over the world. Reversed-phase high-performance liquid chromatography (HPLC) is often used to analyse flavonoids either quantitatively or qualitatively (3 6). However, their separation is still a challenge for analysts because it is difficult to find satisfactory chromatographic conditions (i.e., good resolution with reasonable analysis time). Methanolic extracts from Taxus baccata var. elegantissima and Metasequoia glyptostroboides contain various flavonoid aglycones and glycosides, including dimer forms (biflavonoids) (7 9). Gradient elution is generally recommended for the analysis of flavonoid mixtures. The support of a specialist computer program can facilitate the determination of optimal chromatographic conditions for the separation of analytes of interest. The purpose of this study was, therefore, to optimize the HPLC separation of flavonoids present in methanolic extracts from Taxus baccata var. elegantissima and Metasequoja glyptostroboides. A general HPLC optimization procedure proposed by Snyder (10) was employed. Predictions of gradient conditions for the analysed group of compounds were performed as was an evaluation of simulation reliability. Experimental Sample preparation: Methanolic extracts from leaves of Taxus baccata var. elegantissima and Metasequoja glyptostroboides were prepared as follows. Dried and pulverized plant material (5 g) was extracted in Soxhlet apparatus with 2-naphthyl ether to remove ballast substances (lipophilic compounds, chlorophyll, resins etc.). After drying and removal of ether, plant material was extracted in sequence with chloroform and methanol. From the chloroformic and methanolic extracts solvent was distilled under pressure and the dry residue was dissolved in methanol. In this form it was subjected to chromatographic analysis. The following compounds were dissolved in methanol to prepare 0.1% solutions as reference analytes: quercetin- 7-o-glucoside, luteolin-7-o-glucoside, kaemferol-7-o-glucoside, tricetin-3 -oglucoside, apigenin-7-o-glucoside, quercetin-3-rhamnoside, myricetin, quercetin, kaemferol-3-rhainoside, luteolin, kaemferol, apigenin, cupressuflavone,

2 26 LC GC Europe On-line Supplement Column length Column diameter Table 1: Chromatographic parameters used for computer predictions. 15 cm 0.46 cm Particle size 5.0 µm Pore diameter 10 nm Plate number Temperature Mobile phase Flow-rate Dead time, t 0 Dwell volume, V D 30 C Methanol/phosphate buffer ph 3.0 (v/v) 1.0 ml/min 2.12 min 1.93 ml Retention time, t R (min) Analyte Gradient time Gradient time t G 20 min t G 60 min n. i n. i n. i n. i n. i n. i n. i Quercetin-7-O-glucoside Flavonoid glucoside Flavonoid glucoside Kaemferol-7-O-glucoside n. i Flavonoid glucoside Myricetin Quercetin Amentoflavone Bilobetin O-methylamentoflavone O-methylamentoflavone Ginkgetin Sciadopitysin Table 2 Retention data obtained for methanolic extract from Taxus baccata var. elegantissima in two initial experiments; linear gradient 5-100% B, t G = 20 and 60 min. amentoflavone, bilobetin 4 -omethylamentoflavone, 7-omethylarnentoflavone, ginkgetin, 2,3- dihydrosciadopitysin, sciadopitysin. Chromatographic conditions: Chromatographic measurements were made on a liquid chromatograph (Merck- Hitachi, Frankfurt-Tokyo, Germany-Japan), consisting of a pump (L-7100), diode-array detector (L-7455), autosampler (L-7200), thermostat (L-7350), membrane degasser (L-7612) and interface (D-7000). The column used was an Inertsil ODS-3, cm, packed with octadecyl-bonded silica, particle size 5 µm (GL Sciences Inc., Shinjuku-ku, Tokyo, Japan). The injected sample volume was 20 µl. All chromatographic investigations were performed at 30 C with the flow-rate 1 ml/min. Methanol was obtained from Przedsiebiorstwo Chemiczne Odczynniki Sp z.o.o. Lublin, Poland. Water was prepared with a Milli-Q Water Purification System (Millipore Corp., Bedford, Massachusetts, USA). Phosphate buffer (20 mm, ph 3.0) was prepared by dissolving the appropriate quantity of sodium dihydrogen phosphate dihydrate (Merck KGaA, Darmstadt, Germany) in pure water, and adjusting the ph with hydrochloric acid 37% (Fluka Chemie AG, Buchs, Switzerland). The buffer ph was measured at 21 C before addition of the organic modifier. The measurements were performed with an HI 9017 ph meter (Hanna Instruments, Bedfordshire, UK). HPLC procedure and computer simulations: Chromatographic analysis of the extracts was performed according to the general optimization scheme recommended in reference 10. First, two initial gradient experiments were performed (for t G at 20 and 60 min, and linear gradient 5 100% B, where B is the organic modifier [methanol]). Retention data for all the identified and most of the unidentified analytes from these initial experiments were input data for computer predictions. Computer simulations were performed with DryLab for Windows, version 2.0 (LC Resources, Walnut Creek, California, USA). Attempts were made to choose the best chromatographic conditions (optimal gradient time and

3 27 No. Analyte t Rcalc t Rexpt t Rcalc -t Rexpt δϕ δδϕ 1 n. i n. i n. i n. i n. i n. i n. i Quercetin-7-O-glucoside Flavonoid glucoside Flavonoid glucoside Kaemferol-7-O-glucoside n. i Flavonoid glucoside Myricetin Quercetin Amentoflavone Bilobetin O-methyloamentoflavone O-methyloamentoflavone Ginkgetin Sciadopitysin Mean: Table 3 A comparison of predicted and experimental retention data for a separation of methanolic extract Taxus baccata var. elegantissima; linear gradient 5-100% B, t G 35 min. No. Analyte t Rcalc t Rexpt t Rcalc -t Rexpt δϕ δδϕ 1 Quercetin-7-O-glucoside Flavonoid glucoside Flavonoid glucoside Kaemferol-7-O-glucoside Flavonoid glucoside Myricetin Quercetin Amentoflavone Bilobetin O-methyloamentoflavone O-methyloamentoflavone Ginkgetin Sciadopitysin Mean: Table 4 A comparison of predicted and experimental retention data for a separation of methanolic extract Taxus baccata var. elegantissima; linear gradient 5-100% B, t G 25 min.

4 28 LC GC Europe On-line Supplement Figure 1: HPLC separation of methanolic extract from Taxus baccata var. elegantissima obtained with a linear gradient 5 100% B, tg = 35 min: (a) Predicted chromatogram, gradient range). Under the conditions chosen experiments were executed and the results achieved were compared with those obtained by computer simulations (predicted results). Predictive errors were evaluated according to the procedure reported in reference 11. Errors in predicted values of retention time, t R are expressed in terms of an equivalent change (error),, in the volume fraction of the organic modifier,, for the predicted separation. Therefore, the predicted value of t R (k) for a mobile-phase composition should correspond to the correct value of t R (k) for the mobilephase composition. So, in the first part of the calculation, the experimental and predicted retention factors are compared on the basis of the following equation (11): = t R ( /t G ) [1] where is the error in predicted retention factor expressed as an equivalent change in ; t R is the difference between predicted and experimental retention time; is the volume fraction of the strong solvent in the mobile phase; is the change in w during the gradient and t G is gradient time Figure 2: HPLC separation of methanolic extract from Taxus baccata var. elegantissima obtained with a linear gradient 5 100% B, tg = 25 min: (a) Predicted chromatogram,

5 29 The second part of the calculation uses the relationship between differences. Errors in t R for the adjacent peaks i and j can be expressed as (11): = ( ) j ( ) i [2] where is the error in predicted resolution (R s ) because of the errors in the predicted retention expressed as the equivalent change of ; ( ) j and ( ) i are the values of for adjacent bands i and j. The maximum average value of is 0.001, which corresponds to an average error of 0.2 in R s (acceptable for complex samples if one assumes an average value of column plate number, N = ) (11). Results and Discussion Analysis of Taxus baccata var. elegantissima extract: In the Taxus baccata var. elegantissima extract, 13 flavonoids were identified. Among them, six were flavonoid dimers. Chromatographic process parameters (Table 1) and retention times (Table 2) obtained experimentally from two initial experiments (input data) were entered into the DryLab program. Retention time, t R (min) Analyte Gradient time Gradient time t G 20 min t G 60 min n. i n. i n. i n. i n. i Luteolin-7-O-glucoside n. i Tricetin-3 -O-glukoside Apigenin-7-O-glucoside Quercetin-3-rhamnoside n. i Kaemferol-3-α-rhamnoside n. i Luteolin Kaemferol Apigenin n. i Amentoflavone Bilobetin methylcupressuflavone O-methylamentoflavone n. i O-methylamentoflavone Ginkgetin ,3-dihydrosciadopitysin Sciadopitysin Table 6 Retention data obtained for methanolic extract from Metasequoia glyptostroboides in two initial experiments; linear gradient 5-100% B, t G equal 20 and 60 min. No. Analyte t Rcalc t Rexpt t Rcalc -t Rexpt δϕ δδϕ 1 Quercetin-7-O-glucoside Flavonoid glucoside Flavonoid glucoside Kaemferol-7-O-glucoside Flavonoid glucoside Myricetin Quercetin Amentoflavone Bilobetin O-methyloamentoflavone O-methyloamentoflavone Ginkgetin Sciadopitysin Mean: Table 5 A comparison of predicted and experimental retention data for a separation of methanolic extract Taxus baccata var. elegantissima; linear gradient 38-85% B, t G 12 min.

6 30 LC GC Europe On-line Supplement Figure 3: HPLC separation of methanolic extract from Taxus baccata var. elegantissima obtained with a linear gradient 38 85% B, tg = 12 min: (a) Predicted chromatogram, Satisfactory resolution for 5 100% B linear gradient were found at the gradient time t G = 35 min. The comparison of simulated and experimental chromatograms is shown in Figure 1. Calculated predictive errors ( and ) are collected in Table 3. It can be seen that the predictions are very accurate (average = and average = ). Good resolution for the known flavonoids was achieved using 5 100% B linear gradient at gradient time t G = 25 min. The predicted and experimental retention data are in excellent agreement (Table 4, Figure 2). Average predictive errors were: = and = Because the first known compound eluted at a retention time, t R = min, attempts were made to accelerate the whole separation. Using the software it was found that the best separation for analysed flavonoids would be achieved with a linear gradient of 38 85% B at t G = 20 min (Figure 3(a)). By increasing the initial concentration of methanol (from 5 to 38%), the retention time for the first fiavonoid was shortened to 6.77 min (Figure 3(b)). However, the accuracy of the whole prediction under such conditions appeared to decrease in comparison with the former analysis (Table 5). This could be the result of a big difference in the initial composition of the mobile phase between that gradient (an initial concentration of methanol: 38%) and the gradients used for obtaining the input data (an initial concentration of methanol: 5%). Analysis of Metasequoja glyptostroboides extract: Sixteen flavonoids were identified, among them eight biflavonoids. Similar to the extract from Taxus baccata var. elegantissima, the retention data from two gradient experiments provided input data to create computer predictions (Table 6). General experimental conditions were the same as with Taxus baccata var. elegantissima (Table 1). It was found that for the linear gradient 5 100% B a satisfactory separation results at a gradient time t G = 40 min (Figure 4(a)). Experimental results are shown in Figure 4(b). A comparison of experimental and Figure 4: HPLC separation of methanolic extract from Metasequoia glytostroboides obtained with a linear gradient 5 100% B, tg = 40 min: (a) Predicted chromatogram,

7 31 predicted retention data is illustrated in Table 7. It can be seen that average predictive errors are very small ( = and =0.0004). An attempt to shorten the analysis time was undertaken by simulation of a 30 90% B linear gradient at the gradient time t G = 30 min (Figure 5(a)). The use of a higher concentration of methanol in the initial composition of the mobile phase decreased retention times of all the compounds and effectively shortened the analysis time (Figure 5(b)). However, the predictive errors appeared to increase in comparison with those observed in the analysis performed with 5 100% B gradient (Table 8). Conclusions Optimization of the chromatographic separations of flavonoids from Tarus No. Analyte t Rcalc t Rexpt t Rcalc -t Rexpt δϕ δδϕ 1 n. i n. i n. i n. i n. i Luteolin-7-O-glucoside n. i Tricetin-3 -O-glukoside Apigenin-7-O-glucoside Quercetin-3-rhamnoside n. i Kaemferol-3-α-rhamnoside n. i Luteolin Kaemferol Apigenin n. i Amentoflavone Bilobetin methylcupressuflavone O-methylamentoflavone n. i O-methylamentoflavone Ginkgetin ,3-dihydrosciadopitysin Sciadopitysin Mean: Table 7 A comparison of predicted and experimental retention data for a separation of methanolic extract from Metasequoia glyptostroboides in two initial experiments; linear gradient 5-100% B, t G 40 min.

8 32 LC GC Europe On-line Supplement baccata var. elegantissima and Metasequoia glyptostroboides can be performed with the use of a computer program. Optimizing the chromatographic separation of analytes in a complex mixture can be realized without the need for traditional trial-and-error methods. The use of linear gradients provides good chromatographic separations of flavonoids in accordance with simulations predicted by means of a computer program. The results obtained confirm the unique potential of the optimized gradient approach to separate components of biological samples as has independently been observed by other authors (12, 13) Figure 5: HPLC separation of methanolic extract from Metasequoia glytostroboides obtained with a linear gradient 30 90% B, tg = 30 min: (a) Predicted chromatogram, References (1) H.-J. Wang, and P.A. Murphy, J. Agric. Food Chem., 42 (1994) (2) D.E. Pratt and P.M. Birac, J. Food Sci., 44 (1979) (3) H. Schulz and G. Albroscheit, J. Chromatogr., 442 (1988) (4) O. Sticher, Planta Med., 59 (1993) (5) P. Pietta et al., J. Chromatogr., 553 (1991) (6) M. Keinänen and R. Julkunen-Tutto, J. Chromatogr. A, 793 (1998) (7) P. Pieffa, P. Mauri and A. Rava, J. Chromatogr., 437 (1988) (8) A. Hasler, O. Sticher and B. Meier, J. Chroniatogr., 605 (1992) (9) O. Sticher, Planta Med., 59 (1993) (10) L.R. Snyder, J.J. Kirkland and J.L. Glajch, Practical HPLC Method Development, John Wiley & Sons, New York, (11) J.W. Dolan et al., J. Chromatogr. A, 857 (1999) (12) P.L. Zhu et al., J. Chromatogr. A, 756 (1996) (13) J.W. Dolan et al., J. Chromatogr. A, 803 (1998)

9 33 No. Analit t Rcalc t Rexpt t Rcalc -t Rexpt δϕ δδϕ 1 n. i n. i n. i n. i n. i Luteolin-7-O-glucoside n. i Tricetin-3 -O-glukoside Apigenin-7-O-rhamnoside Quercetin-3-rhamnoside n. i Kaemferol-3-α-rhamnoside n. i Luteolin Kaemferol Apigenin n. i Amentoflavone Bilobetin O-methylcupressuflavone O-methylamentoflavone n. i O-methylamentoflavone Ginkgetin ,3-dihdrosciadopitysin Sciadopitysin Mean: Table 8 A comparison of predicted and experimental retention data for a separation of methanolic extract from Metasequoia glyptostroboides; linear gradient 30-90% B, t G 30 min.

RITONAVIRI COMPRESSI RITONAVIR TABLETS. Final text for addition to The International Pharmacopoeia (July 2012)

July 2012 RITONAVIRI COMPRESSI RITONAVIR TABLETS Final text for addition to The International Pharmacopoeia (July 2012) This monograph was adopted at the Forty-sixth WHO Expert Committee on Specifications