Field-amplified sample stacking–sweeping of vitamins B determination in capillary electrophoresis

Journal of Chromatography A, 1267 (2012) 224–230

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Field-amplified sample stacking–sweeping of vitamins B determination in capillary electrophoresis ˛ Szymon Dziomba, Piotr Kowalski ∗ , Tomasz Baczek Department of Pharmaceutical Chemistry, Medical University of Gda´ nsk, Hallera 107, 80-416 Gda´ nsk, Poland

a r t i c l e

i n f o

Article history: Available online 28 July 2012 Keywords: Field-amplified sample stacking (FASS) Sweeping Vitamins B Bacterial growth medium Ilex paraguariensis

a b s t r a c t A capillary electrophoretic method for determination of five water soluble vitamins B along with baclofen as an internal standard has been developed and assessed in context of precision, accuracy, sensitivity, freedom from interference, linearity, detection and quantification limits. On-line preconcentration technique, namely field-amplified sample stacking (FASS)–sweeping, has been employed in respect to obtain more sensitive analysis. Separation conditions received after optimization procedure were as following background electrolyte (BGE), 10 mM NaH2 PO4 , 80 mM SDS, (pH 7.25); sample matrix (SM), 10 mM NaH2 PO4 (pH 4.60); uncoated fused silica capillary (50 ␮m i.d. × 67 cm length); UV spectrophotometric detection at 200 nm; injection times: 10 s and 30 s at 3.45 kPa; applied voltage 22 kV; temperature 22 ◦ C. Validation parameters, namely precision, accuracy and linearity, were considered as satisfactory. Under the optimized conditions, it has been also successfully applied for vitamins B determination in bacterial growth medium and commercially available Ilex paraguariensis leaves. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Discovered at the beginning of last century, vitamins B have become one of the main goal of interest for nutrition, biochemistry and many other science. Their participation in the biochemical processes has been thoroughly examined which results in better understanding of life cycles. These researches not only allowed to prevent some ailments like beriberi or pellagra but also microorganisms biochemistry engineering. This is especially important for the biotechnology industry, which is next to the chemical synthesis main vitamins producer. It is estimated that world production is about thousands of tons per year of each vitamin. It is due to the high demand by the pharmaceutical and food market. It is clear that researches on vitamins B need simultaneous reliable and sensitive analytical methods. However, their simultaneous determination is often impossible without previous laborand time-consuming separation of the analytes from very complex sample matrix. Thus, high-performance separation techniques seem to be ideal for its quantification. Most of developed methods based on capillary electrophoresis (CE) and high- and ultra-high performance liquid chromatography (HPLC and UPLC, respectively) concerns recently mainly on the assessment of vitamin B levels in pharmaceutical products and food samples due to the relatively high concentration of analytes [1–5]. Advantage of those chromatographic techniques is associated with the possibility to obtain lower

∗ Corresponding author. Tel.: +48 58 349 31 36. E-mail addresses: [email protected], [email protected] (P. Kowalski). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.07.068

detection limits due to the larger volume of the injected sample and larger capillary inner diameter in comparison to CE. Because sensitivity is repeatedly very important, particularly when biological samples are tested, chromatographic techniques seem to be better choice. However, CE techniques have one superiority over chromatographic ones- a great efficiency, which in some of the analyses is an invaluable feature. Moreover, the sensitivity in CE can be improved using powerful detectors, like a laser-induced fluorescence (LIF), which however for vitamin determination can be used only for riboflavin quantification [6]. Likewise, to increase the sensitivity of the method, various types of extraction to enrich the analytes in the sample are employed [7,8]. Unfortunately, these methods are usually time-consuming and have a risk of loss of analytes during sample preparation procedures. On-line preconcentration techniques do not have these disadvantages and can be successfully used for electrophoretic separation, immediately after parameters optimization procedure [9]. Furthermore, the possibility of coupling the CE technique with high class detectors makes them a valuable analytical tool. In the last 15 years, many on-line preconcentration techniques have been also reported [10–12]. Most of them base on stacking [13–15], sweeping [16,17], dynamic pH junction [18,19] and isotachophoresis phenomenon [20–22]. Researches in this field have shown that employment of electrokinetic injection provides stronger signal improvement than hydrodynamic one. This is a result of combination of electroosmotic and electrophoretic injection mechanisms. Additionally, hydrodynamic injection-based preconcentration is strongly limited by diffusion processes occurring in the capillary when larger sample volumes

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plugs are employed. Unfortunately, the use of electrokinetic injection is efficient only if the chemical structures of analytes allow for this, namely when ionization of analytes in the sample matrix is not completely different (positive and negative ions). If it is not possible, the hydrodynamic injection technique can be employed. In those cases, coupling of preconcentration techniques like dynamic pH junction-sweeping [23], micelle to solvent stacking–sweeping [24] or field-amplified sample stacking with dynamic pH junction [25] can provide better final results. In the paper, the CE method for simultaneous determination of five water soluble vitamins using FASS–sweeping technique was optimized. To the best of our knowledge, this is the first report of combination of FASS–sweeping technique for determination of varied compounds in micellar electrokinetic chromatography [26]. The elaborated method validation parameters have been designed and the method was successfully applied to the determination of these compounds in bacterial growth media and cooperatively Ilex paraguariensis leaves. Enhancement factors obtained in the study were about up to 40-fold in comparison to capillary zone electrophoresis (CZE) technique, using standard hydrodynamic plug (2 s, 0.5 psi).

NH2

All electrophoretic experiments were performed on P/ACE 5000 CE system (Beckman Instruments, Fullerton, CA, USA) equipped with UV detector. Uncoated fused-silica capillary dimensions were 67 cm × 50 ␮m i.d. (Beckman); temperature was controlled at 22 ◦ C. UV detection was performed at 200 nm. Buffer pH values were measured with Cerko Lab System pH-meter (Gdansk, Poland). Conductivity was measured with Conductibity Meter Lab 970 (SCHOTT Instruments, Mainz, Germany). 2.2. Reagents and solution Sodium dodecyl sulfate (SDS; >98.5%), sodium dihydrogen phosphate dehydrate (>99%) and sodium hydroxide (>98%) were purchased from Sigma Aldrich (St. Louise, MA, USA). Their stock solutions were prepared by dilution of the appropriate amount of substance in redistilled water (GFL 2104, Burgwedel, Germany) to concentration of 200, 100 and 100 mM, respectively. The vitamins B (analytical grade, Sigma Aldrich) stock solution were prepared each day by dilution in redistilled water (thiamine hydrochloride – B1 , nicotinamide – B3 , pyridoxine – B6 ), or in 10 mM sodium tetraborate (folic acid – B9 , riboflavin – B2 ). Chemical structures of the analyzed vitamins were depicted in Fig. 1. The optimized background electrolyte (BGE) was composed of 80 mM SDS and 10 mM NaH2 PO4 , whereas the sample matrix (SM) was 10 mM NaH2 PO4 . Measured pH values of BGE and SM were 7.25 and 4.60, respectively. Desired pH values of prepared buffers solutions were obtained with 1 M NaOH (POCh, Gliwice, Poland) and 85% H3 PO4 (POCh, Gliwice, Poland). Bacteria used in experiments were Staphylococcus aureus (ATCC6538) and Escherichia coli (ATCC8739) strains. Bacterial growth medium used in experiments was BD Mueller Hinton II Broth (Cation Adjusted) prepared to a following final composition: beef extract (3.0 g/L), acid hydrolysate of casein (17.5 g/L), starch (1.5 g/L). Yerba mate was bought in a local shop.

OH

OH

N

N

+

OH

OH

S

N

N

Thiamine (B1) pKa=4.9

Pyridoxine (B6) pKa=5.0

O

N

NH

O

O

N

N

NH2

OH

N

OH

HO

OH

Nicotinamide (B3) pKa=3.35

Riboflavin (B2) pKa=10.2 O

N

N H2 N

N

O N H

OH

2. Experimental 2.1. Apparatus

225

N H

OH OH O

N

Folic acid (B9) pKa1=3.5 pKa=4.3 Fig. 1. Chemical structure and pKa values of analyzed vitamins.

and incubated with E. coli and S. aureus inoculum for 24 h (about 109 CFU/mL). Free growth media has also been incubated as the reference. After incubation medium was filtrated (0.22 ␮m filter pores size), supernatant collected to Eppendorf tubes and stored in freezer (−20 ◦ C). Before CE analysis, samples were centrifuged (14,000 rpm, 5 min) and diluted with NaH2 PO4 to its final concentration of 10 mM. Next, 100 ␮L of such a prepared sample was fortified with 1 ␮L of I.S. (baclofen at 100 ␮g/mL) and analyzed with CE. 2.3.2. I. paraguariensis (yerba mate) leaves 0.1 g of mate leaves was suspended in 50 mL of water and extracted with magnetic stirrer at 1000 rpm for 45 min. After extraction step, water solution was centrifuged (14,000 rpm, 5 min) and diluted 2.5-fold with NaH2 PO4 solution to the final concentration of 10 mM. Subsequently, 100 ␮L of such prepared sample was fortified with 1 ␮L of I.S. (baclofen at 100 ␮g/mL) and analyzed with CE. 2.4. General electrophoresis procedure At the beginning of each day, capillary was conditioned at the pressure of 20 psi (137.9 kPa) consequently with methanol (20 min), 0.1 M NaOH (10 min), redistilled water (10 min) and finally with BGE (5 min). All analyses were performed at 22 kV with normal polarity mode. Between each run, the capillary was flushed with 0.1 M NaOH (2 min), water (2 min) and BGE (1.5 min). Hydrodynamic injection was performed at 0.5 psi (3.45 kPa). Under optimized conditions sample was introduced into the capillary by hydrodynamic injection mode (10 s and 30 s, 0.5 psi). 3. Results and discussion

2.3. Sample preparation 3.1. Separation and FASS–sweeping mechanism 2.3.1. Bacterial growth medium Bacterial growth medium was prepared by dissolving a powder in 1 L of hot water and rapid boiling followed by sterilization

Vitamins B, in terms of chemical structure and properties, are very varied group. Differences essential for efficient electrophoretic

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Fig. 2. Scheme of field-amplified sample stacking–sweeping mechanism. (a) Sample is introduced to the capillary filled with background electrolyte (BGE) containing anionic micelles having higher pH value than low conductivity sample matrix (SM). (b) Application of the voltage cause stacking of charged analytes in the zones boundaries and entering of BGE from the anodic end to capillary with electroosmotic flow (EOF). Penetration of the micelles in low conductivity SM results in sweeping of sample compounds. (e) After separation analytes are detected. Migration velocities are dependent on their charge and affinity to pseudostationary phase.  − – Anionic analyte, ⊕ – cationic analyte,  – non-ionic analyte, – analyte surrounded by micellar envelope.

separation pertain mainly to ionic radius and pKa value of each analyte. On the one hand, variety of these properties can facilitate optimization of separation conditions; on the other hand, choice of proper on-line preconcentration technique is much more difficult. Best results are obtained when using property characteristic for each analyte, e.g., ionization state. In the case of the vitamins B, this is not possible because of their variety (see Fig. 1). In order to solve problems concerning the determination of vitamins B, a field-amplified sample stacking–sweeping technique has been employed. The use of hydrodynamic injection is necessary due to the fact that it is not possible to obtain the same ionization (positive or negative) for all compounds of interest in the sample matrix by its pH value change, which would allow the possibility of electrokinetic injection usage. Schematic mechanism of separation is shown in Fig. 2. At the beginning, the sample was introduced into the capillary using pressure (Fig. 2A). After high voltage application (Fig. 2B), charged analytes stacked on the boundary between BGE and sample matrix (SM) zones. Under the influence of an applied voltage, background electrolyte containing micelles, was introduced into the capillary from the inlet end due to the presence of electroosmotic flow (EOF), and swept the sample zone compounds (Fig. 2C). Thus, stacking mechanism applied only to charged analytes, while sweeping preconcentrated both charged and neutral molecules. When all analytes became swept (Fig. 2C and D) separation occurred (Fig. 2E). Its mechanism was based on micellar electrokinetic chromatography. When anionic surfactant was used as a pseudostationary phase, all analytes gained a net negative charge. The order of detection was seen in Fig. 2E. The longest migration time observed in the case of positively charged analytes is caused by strong solvation of the analyte with micelles. Strong interactions between determined molecules and pseudostationary phase excuse a long migration time. The order in which negatively and non-charged analytes leave the capillary

Fig. 3. Influence of sample matrix ionic strength on stacking of analytes. Conditions; BGE: 80 mM SDS, 10 mM NaH2 PO4 , pH 7.25; SM: (A) water, (B) 10 mM NaH2 PO4 , (C) 20 mM NaH2 PO4 , (D) 40 mM NaH2 PO4 , (E) 60 mM NaH2 PO4 , pH 4.60; uncoated fused silica capillary (50 ␮m i.d. × 67 cm length); UV spectrophotometric detection at 200 nm; injection time 30 s at 3.45 kPa; applied voltage 22 kV; temperature 22 ◦ C.

depends in that way on their size, total ionization and pseudostationary phase affinity. 3.2. Sample matrix ionic strength Under the optimized separation conditions (pH value of micellar BGE), electrolyte concentration in the sample zone has been studied. Stacking phenomenon occurred due to the sudden electrophoretic velocity change of the analyte. It can be caused by the differences in electric field strength between zones during separation. Different concentrations of NaH2 PO4 in SM (ranging from 0 to 60 mM) were tested and its influence on signals amplification can be seen in Fig. 3. The highest difference in the electric field strength is shown in Fig. 3A, where the analytes were dissolved in pure water. As it can be seen, the best results for folic acid (B9 ) signal were obtained with pure water as the sample media. In turn, addition of electrolyte to the sample matrix resulted in deterioration of the peak. On the other hand, the signals for other vitamins have been improved by using 10 mM NaH2 PO4 as SM. Because this concentration have not broadened folic acid signal significantly, it was considered as an optimal. Measured conductivities of the SM and BGE were 0.44 and 1.90 mS/cm (25 ◦ C), respectively.

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3.3. SDS concentration To determine the impact of the amounts of surfactant on the effect of sweeping efficiency, a series of electrophoretic separation with phosphate buffer and SDS at different concentrations were tested (Fig. S1 – Supplementary data). Different SDS concentration has dual effect on the vitamins electrophoresis. First of all, it affected the sweeping of analytes which manifested itself in peaks height and separation efficiency (Fig. S1A–C). Too low concentration of SDS in the BGE can cause all peaks broadening. Particularly noticeable is thiamine (B1 ) peak tailing which effect has been stopped at 80 mM SDS concentration (Fig. S1D), therefore this SDS concentration was considered as optimal. Moreover, greater allowance of SDS caused base line undulation in electrophoretic view (Fig. S1E). Furthermore, the level of SDS has a significant effect on migration times of analytes. Tests showed that the migration times of compounds of interest differed, depending on the increasing concentration of SDS in the same pH buffer value, especially for the riboflavin (B2 ), which displayed a difference of up to 2 min in the range of 60 mM SDS concentration (Fig. S1A–D). Further increase in SDS levels did not cause this effect (Fig. S1D and E). 3.4. Injection time Injection time has great influence on signal amplification (Fig. 4). As it can be seen (Fig. 4A–D), there is a close relationship between peak height and dosing time. With a longer injection time, band broadening of signals was also observed, which resulted with the decreasing of separation efficiency. Moreover, undesired phenomenon of peak shape deformation occurred when 40 s injection time was employed (Fig. 4D). This can be observed in zooms attached to Fig. 4C and D. Such deviations should also be included in method optimization, because it can be a potential source of mistakes obtained during validation process. Due to these facts, one considered that 30 s injection time can be optimal as the strongest signal amplification and 10 s injection can be the compromise between the signal amplification and separation efficiency. 3.5. Sample matrix pH value The influence of different pH values in sample matrix on signals intensities has been also investigated (Fig. S2). For this purpose, one tested several matrix samples containing the same analytes, but diversifying only in pH values (in the range from 3.0 to 9.2). The scope of study included also isohydric pH value, like with background electrolyte at 7.25. The most significant changes in peak height and width can be noticed for folic acid (B9 ) with the best final results when pH values of SM were 7.25 and 9.20. Unfortunately, at these pH values, riboflavin (B2 ) peak broadening has been also observed. Due to the obtained results, one considered pH 4.60 to be optimal for SM. 3.6. Validation parameters study Investigation of injection time has shown that the greatest signal amplification has been obtained for 30 s, which provides about 1.5- to 3-fold better result in comparison with 10 s injection time. Unfortunately, due to the lower separation efficiency, 10 s injection time method is more suitable for highly complex matrices analysis. According to these facts, both methods (10 s and 30 s) validation parameters have been designated and applied in this paper. Validation parameters obtained during the study are presented in Tables 1 and 2. The CE method was evaluated in compliance with the analytical performance parameters required for analytical method validation, including sensitivity, freedom from

Fig. 4. Influence of injection time on peaks shape and intensity. Conditions; BGE: 80 mM SDS, 10 mM NaH2 PO4 , pH 7.25; SM: 10 mM NaH2 PO4 , pH 4.60; injection time: (A) 10 s, (B) 20 s, (C) 30 s, and (D) 40 s. Other conditions are the same as in Fig. 3.

interference, precision, accuracy, linearity, detection and quantification limits. The experiments were carried out on three consecutive days and main parameters were determined for samples spiked with three different concentrations. Standard calibration curves were prepared by the injection of mixed-standard solutions on six concentration levels (1.0, 2.0, 5.0, and 10.0 ␮g/mL and two lower levels, different for each vitamin and injection time method). As it can be noticed there are some differences between 10 and 30 s injection time methods. Longer injection plug has improved the sensitivity of all analytes for about 1.5–3 times allowing to obtain detection limits in the range of 0.05–0.1 ␮g/mL for longer injection time method and 0.1–0.3 ␮g/mL for shorter injection time method. Both methods validation parameters have been designed for vitamins B of interest quantification from several 100 ng/mL to 10 ␮g/mL range. All correlation coefficients obtained were above 0.9990 and 0.9993 for 10 s and 30 s methods, respectively. Validation parameters values obtained for six concentration points using peak heights were satisfactory and below 9.1% (for precision expressed as %RSD) and 1.08% (for corrected migration times expressed as %RSD), and in the range of 88.6–105.6% (for

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Table 1 Quality parameters obtained for the validation of investigated vitamins B with the use of 10 s and 30 s injection method. Validation parameters 30 s injection time Linearity range (␮g/mL) Regression equation R2 LOD (␮g/mL) 10 s injection time Linearity range (␮g/mL) Regression equation R2 LOD (␮g/mL) CZE LOD (␮g/mL)

B1

B2

B3

B6

B9

0.25–10 y = 0.425x + 0.078 0.9993 0.05

0.35–10 y = 0.193x + 0.015 0.9997 0.10

0.25–10 y = 0.209x + 0.022 0.9998 0.08

0.25–10 y = 0.225x + 0.052 0.9996 0.08

0.25–10 y = 0.364x + 0.036 0.9994 0.07

0.3–10 y = 0.334x + 0.047 0.9999 0.10

0.9–10 y = 0.148x + 0.025 0.9997 0.3

0.5–10 y = 0.247x + 0.052 0.9990 0.15

0.4–10 y = 0.183x + 0.060 0.9998 0.15

0.4–10 y = 0.334x + 0.016 0.9996 0.12

2.00

2.00

1.00

1.00

1.00

Table 2 Results obtained for inter- and intra-day accuracy (Acc, %) and precision (Pre, %RSD). Vit

C

10 s

30 s

Intra-day

B1

B2

B3

B6

B9 a

2 1 0.5 2 1 0.5 2 1 0.5 2 1 0.5 2 1 0.5

Inter-day

Intra-day

Inter-day

Acc

Pre

Acc

Pre

Acc

Pre

Acc

Pre

99.9 104.3 92.7 103.5 96.3 101.7a 103.4 97.4 91.5 104.9 103.1 89.2 99.9 98.6 98.0

3.19 3.88 4.91 4.14 4.31 7.44a 2.78 3.81 5.26 2.58 3.13 4.88 5.31 2.47 6.61

97.6 104.8 93.2 104.9 95.2 103.6a 105.0 94.2 89.2 105.6 103.9 88.8 92.9 94.3 95.7

4.63 5.12 7.60 5.90 6.52 8.98a 4.63 5.02 7.15 4.09 5.54 6.90 6.12 7.36 8.00

104.0 97.4 89.1 94.4 101.4 103.6 104.5 94.3 96.0 104.6 103.3 97.7 94.8 93.7 103.7

5.05 4.58 5.26 3.22 5.65 5.66 5.14 4.80 8.13 3.22 3.06 3.94 5.90 5.92 6.89

104.5 96.1 88.6 95.5 103.1 103.9 105.2 93.8 95.2 104.8 104.4 95.8 93.8 92.1 104.9

6.98 7.53 8.18 6.02 7.85 8.50 5.95 6.40 8.65 3.60 4.96 5.18 6.45 7.37 8.08

Data for concentration 0.9 ␮g/mL.

accuracy). Detection limits obtained for 30 s injection time method have been compared with LODs obtained with CZE method (BGE, 60 mM NaH2 PO4 ; injection time, 2 s; other conditions as in Fig. 3B). LODs have been designated separately for each vitamin with CZE method. Enhancement factors obtained for FASS–sweeping method in comparison to CZE method were about 12 (B3 ), 12 (B6 ), 14 (B9 ), 20 (B2 ), and 40 (B1 ). 3.7. Application 3.7.1. Bacterial growth medium Vitamins B concentration in standard bacterial growth media before and after incubation has been investigated. After 30-fold dilution with water and NaH2 PO4 solution to phosphate final concentration of 10 mM, the samples were directly analyzed by CE. Bacterial growth media are very complex mixture of organic and inorganic compounds, affecting its conductivity and viscosity. Therefore, elimination of matrix influence on physical properties of analyzed samples was necessary. Moreover, dilution of the samples also reduced strong background effect in the form of high baseline absorbance. A typical electropherogram of free growth media sample can be seen in Fig. 5. Due to the complexity of these samples, 10 s injection has been used to achieve high separation efficiency. It should be noticed that baclofen (used as internal standard) signal has been completely separated from other sample compounds. Comparison of electropherograms obtained from growth medium analysis is presented in Fig. 6A and B. Under optimized separation parameters, four vitamins B (B1 , B2 , B3 , and B6 ) in investigated samples have been detected. The identification of analytes has been confirmed on the basis of the corrected migration times and the

Fig. 5. Exemplary electropherogram of bacterial free growth medium. Conditions; BGE: 80 mM SDS, 10 mM NaH2 PO4 , pH 7.25; SM: 10 mM NaH2 PO4 , pH 4.60. Other conditions are the same as in Fig. 3.

standard addition method. Under the elaborated conditions, the folic acid (B9 ) concentration has been below its detection limit, while the concentration of nicotinamide (B3 ) allowed quantifying it (Table 3). Decrease of the nicotinamide content in medium during Table 3 Results obtained in analysis of commercially available Ilex paraguariensis leafs (data in ␮g/g) and bacterial growth medium before and after incubation with S. aureus and E. coli (data in ␮g/mL). Sample

B3

B6

B9

B2

B1

Free growth medium S. aureus E. coli Yerba mate

51.1 41.9 33.6 LOD>

LOQ> LOQ> LOQ> 429

LOD> LOD> LOD> LOD>

LOQ> LOQ> LOQ> LOD>

LOQ> LOQ> LOQ> LOD>

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229

excess in this process provides high extraction efficiency. Under these circumstances, the analytes can occurred in extract at low concentration. Due to this fact it is advisable to use on-line preconcentration techniques. In this paper we investigated vitamins in commercially available I. paraguariensis leaves also known as yerba mate. Samples were prepared as it was described in Section 2.3.2 and have been analyzed using CE technique under optimized conditions employing 30 s injection time. A typical electropherogram obtained from mate water extract sample can be seen in Fig. 7. The estimated content of pyridoxine (B6 ) in mate leaves was 429 ␮g/g. 4. Conclusions

Fig. 6. Comparison of electropherograms obtained from analysis of (A) vitamins B3 and B6 and (B) vitamins B1 and B2 in bacterial growth medium before (red solid line) and after incubation with S. aureus (blue dashed line) and E. coli (black dotted line). Electrophoretic conditions are the same as in Fig. 5. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

incubation has been noticed. Moreover, although vitamins B1 and B2 concentrations were too low to quantify them with good precision, decrease in signal intensity after their incubation can also be seen. In the summary, the elaborated method can be used for monitoring of vitamins B in bacterial growth medium. Although some vitamins concentration was too low for quantification, the method can be still employed in industrial biosynthesis with the use of highyield microorganisms for substrates and products concentration monitoring [27]. 3.7.2. I. paraguariensis (yerba mate) leaves Quantification of chemical compounds in plant material requires usually previous extraction process. Usage of solvent

Fig. 7. Electropherogram obtained from analysis of water extracts of commercially available Ilex paraguariensis leaves. Electrophoretic conditions are the same as in Fig. 5.

On-line preconcentration CE method based on complex mechanism (field-amplified sample stacking–sweeping) has been developed. Under the optimization process such crucial factors were included, like sample matrix ionic strength, SDS concentration in the BGE, injection times and SM pH value. Moreover, theoretical aspects of presented techniques have been also discussed. The CE method validation parameters were designed for 10 and 30 s injection time with good accuracy, precision and linearity. The elaborated method for both injection times has been successfully applied for vitamins B quantification in standard bacterial growth medium as well as in commercially available I. paraguariensis leaves. The differences in concentration of nicotinamide in investigated samples before and after incubation with different bacteria species have been presented. Enhancement factors obtained for quantified vitamins B are up to 40-fold and can be considered as typical for stacking and sweeping techniques of relatively hydrophilic compounds [21,23,13,28,29]. A new approach to the determination of small molecules by CE with FASS–sweeping, as mode of preconcentration, has been shown. The presented CE method can be successfully applied to monitor vitamins B level in industrial bioreactors as well as to estimate their content in plant materials. Acknowledgments Authors are grateful to Dr. Rafał Hałasa for the technical support during the preparation of bacterial growth medium. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma. 2012.07.068. References [1] J. Zhang, U. Chakraborty, J.P. Foley, Electrophoresis 30 (2009) 3971. [2] R. Hau Fung Cheung, J.G. Hughes, P.J. Marriott, D.M. Small, Food Chem. 112 (2009) 507. [3] R. Hau Fung Cheung, P.D. Morrison, D.M. Small, P.J. Marriott, J. Chromatogr. A 1213 (2008). [4] B.J. Petteys, E.L. Frank, Clin. Chim. Acta 412 (2011) 38. [5] M. Ciulu, S. Solinas, I. Floris, A. Panzanelli, M.I. Pilo, P.C. Piu, N. Spano, G. Sanna, Talanta 83 (2011) 924. [6] L. Hu, X. Yang, C. Wang, H. Yuan, D. Xiao, J. Chromatogr. B 856 (2007) 245. [7] B.B. Prasad, K. Tiwari, M. Singh, P.S. Sharma, A.K. Patel, S. Srivastava, J. Chromatogr. A 1198–1199 (2008) 59. ˜ I. López-García, M. Bravo, M. Hernández-Córdoba, Anal. Bioanal. Chem. [8] P. Vinas, 401 (2011) 1393. [9] Q. Liu, Y. Liu, Y. Guan, L. Jia, J. Sep. Sci. 32 (2009) 1011. [10] S.L. Simpson, J.P. Quirino, S. Terabe, J. Chromatogr. A 1184 (2008) 504. [11] M.C. Breadmore, J.R.E. Thabano, M. Dawod, A.A. Kazarian, J.P. Quirino, R.M. Guijt, Electrophoresis 30 (2009) 230. [12] M.C. Breadmore, M. Dawod, J.P. Quirino, Electrophoresis 32 (2010) 127. [13] J.P. Quirino, J. Chromatogr. A 1216 (2009) 294. [14] J. Quirino, S. Terabe, Anal. Chem. 72 (2000) 1023. [15] J.P. Quirino, P.R. Haddad, Anal. Chem. 80 (2008) 6824. [16] X. Zhang, Z. Zhang, J. Pharm. Biomed. Anal. 56 (2011) 330.

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