Reproducibility of sample separation using liquid or forced air convection thermostatted high performance capillary electrophoresis systems

Run-to-run sample separation reproducibility has been compared on two commercial high performance capillary electrophoresis units which differ in the mode by which the capillary temperature is thermostatted. Three standard analytes, differing dramatically in molecular character and size, were used for the analysis: benzoic acid, a 14 amino acid peptide from human chorionic gonadotropin, and ribonuclease A represent, respectively, small stable organic molecules, small peptides with little or no secondary structure, and proteins with secondary structure. These standards were evaluated with regard to reproducibility of migration time, peak area, and peak height. The analyses, performed in buffers of optimum pH for the separations, demonstrated that the liquid and forced air convection thermostatted systems both performed extremely well. The reproducibility, as judged by the percent coefficient of variance (% CV) of replicate analyses, was generally found to be less than 1 % (migration time); the reproducibility decreased in the order migration time > peak height > peak area. Whereas the absolute % CV values for MT_rel (migration relative to a standard) observed with the liquid thermostatted system were 2- to 4-fold lower than those observed with the forced air convection thermostatted system, there was little statistically significant difference between the two. As expected, the data indicated a reduction in reproducibility as the complexity of the analyte increased, perhaps as the result of an increased potential for wall interactions. Comparing separations in which low (≈?1 watt/meter [W/m] of capillary) and high (>5 W/m) Joule heat was generated by altering the sodium chloride content of the buffer revealed few statistically significant differences in the reproducibility obtained from the two systems. With these particular standard analytes and their respective buffer systems, there appears to be little difference between forced air convection and liquid thermostatting of the capillary.     

Improved repeatability and matrix-assisted desorption/ionization - time of flight mass spectrometry compatibility in capillary isoelectric focusing

Low repeatability of migration time, peak area, and linearity (pI vs. mobilization time) is a problem often encountered in capillary IEF (cIEF) and is mainly caused by protein precipitation and protein-wall interactions. In order to study the influence of these phenomena, the effect of different classes of additives on repeatability of migration time, peak area and linearity of a mixture of seven model proteins has been investigated. Moreover, the influence of these additives on protein signal suppression in MALDI-TOF MS has been studied. The optimal ampholyte blend (stabilizes pH gradient) to be used depends on the selected UV detection wavelength. All tested ampholyte blends show a significant and comparable signal suppression in MS. The best detergent (to prevent precipitation and wall interaction) should be determined for each sample individually, but generally polyethylene oxide and zwitterionic detergents show good repeatability for migration time (RSD <4.5%) and peak area (majority <10%). The RSD of R(2) is <1.3% for the hydrophilic protein mixture. However, these components cause severe signal suppression in MS. Therefore glucoside detergents should preferably be used for MS coupling. Viscosity-increasing agents (for hydrodynamic wall coating and to minimize diffusion) in particular cellulose derivatives, give good repeatability for migration times (RSD <4.5% at lower concentrations), peak area (except for high concentration methylcellulose and hydroxyethylcellulose all within 7.5%), and correlation (pI vs. migration time), but severe signal suppression is observed in MALDI-TOF MS. Overall, cIEF repeatability and linearity can significantly be improved by adding the appropriate components. However, when the system is coupled to a MALDI-TOF MS, compromises have to be made between high repeatability and linearity on one hand and MS signal intensity on the other.     

Simultaneous analysis of polyhydroxylated alkaloids by capillary electrophoresis using borate complexation and evaluation of sweeping technique for sensitivity improvement

Summary Capillary zone electrophoresis coupled to UV detection was used for the simultaneous analysis of naturally occurring polyhydroxylated alkaloids. This separation was based on anin-situ complexation with borate ions. The effect of parameters such as borate concentration, capillary temperature and analyte molecular structure on migration times and selectivity were discussed. The best separation was obtained with a fused silica capillary (48.5 cm total length ×50 μm I.D., with a bubble factor of 3), 80 mM sodium tetraborate aqueous solution at pH 9.2 and temperature of 20°C. The method was validated and showed good data in terms of migration time and peak area reproducibility, selectivity, linearity and accuracy. The validated method was applied to determine miglitol in commercially available pharmaceutical tablets. To further improve method sensitivity, a sweeping technique involving borate ions was evaluated. This technique was found very sensitive to the analyte complexation with borate, borate concentration, and temperature as well as sample matrix. In the case of miglitol, a 35-fold improvement in peak height was achieved.     

Influence of Counter Pressure on Separation Performance in SDS-MEKC

The influence of a hydrostatic counter pressure on basic mobility equations in MEKC was analyzed theoretically and investigated experimentally. Taking into account the influence of counter pressure, the modified mobility equations predict a decreased migration time ratio t₀/t_MC, which was confirmed by results obtained in SDS-MEKC separation of a homologous series of n-alkyl phenyl ketones. As a result, the migration time window could be enlarged in a simple way without having to change the chemistry of the system. Although counter pressures reduce peak efficiency in MEKC by disturbing the plug profile, the concomitant enlargement of the migration time window compensates for this effect. Especially for later migrating compounds, peak resolution could be enhanced at moderate counter pressures. The overall peak capacities within the whole migration window increases slightly when applying counter pressures up to 10 mbar.     

Paper‐based solid‐phase microextraction for analysis of 8‐hydroxy‐2’‐deoxyguanosine in urine sample by CE‐LIF

An easy‐to‐do paper‐based solid‐phase microextraction (p‐SPME) was developed for determination of 8‐hydroxy‐2’‐deoxyguanosine (8‐OHdG) in urine sample by CE‐LIF. Small piece of filter paper was used as a solid phase to extract 8‐OHdG from urine sample. Its primary mechanism is based on the hydrogen‐bonding interaction between 8‐OHdG and cellulose molecules. The effects of the pH of the sample solution, extraction time, and temperature on the peak area of the analyte were investigated in order to obtain the optimal p‐SPME conditions. Comparing with the untreated sample, the p‐SPME can significantly reduce the interference to the separation of 8‐OHdG by CE‐LIF. Meanwhile, the p‐SPEM can provide more than three times concentrated effect. The developed method was evaluated according to an FDA guideline for biological analysis. The precisions (RSD%, n = 5) of the peak area and migration time of the analyte at three different concentrations were within 3.02–5.82% and 0.92–1.58%, respectively. The limit of identification of the method is about 5 nM according to the significant difference between two sets of the samples with and without spiking the standard (Student's t ‐test, p < 0.05). Good linearity was obtained in the range of 10–1000 nM (R ²>0.99) based on the standard addition. The recoveries at three different concentrations were within 99.8–103.5%. The results of the real sample analysis are consistent with those reported in our previous paper (Electrophoresis 2014, 35, 1873–1879).     

Optimization and Validation of a Capillary MEKC Method for Determination of Proteins in Urine

Proteinuria, i.e. increased excretion of proteins in urine, is a common sign indicating renal or urinary tract diseases. In this study, a fast and simple procedure for urine sample preparation and capillary micellar electrokinetic chromatographic analysis is presented, without any sample pretreatment prior to the analysis. The developed MEKC method was employed for simultaneous determination of albumin (ALB), haemoglobin (HGB), and myoglobin (MYO) in human urine samples obtained from patients with diagnosed proteinuria. Optimum conditions for detection and separation of ALB, HGB, and MYO are 50 mmol L⁻¹ borate buffer containing 20 mmol L⁻¹ SDS (pH 9.3), injection 40 mbar × 20 s, voltage 25 kV, temperature 30 °C, and detection wavelength 200 nm. The method was shown to be specific, accurate, linear (correlation coefficients r ² > 0.99), and precise (RSD below 3.75 and 7.23% for migration time and peak area, respectively). Multi-variable-at-a-time (MVAT) approach for robustness testing shows no significant variations in accuracy, specificity, and precision as RSD values were lower than 5 and 10% for migration time and peak area, respectively. The presented method is applicable for routine analyses of urine samples as a screening method for patients with excess ALB, HGB, and MYO.

     

高效毛细管区带电泳二肽类组分迁移行为的研究

本文考察了影响二肽类组分在高效毛细管区带电泳上迁移时间重复性的因素,提出了采用相对指标来作为高效毛细管区带电泳峰定性的依据,并考察了电流以及柱温对迁移时间和相对迁移时间的影响,发现在恒压lgt~m与T^-1有良好的线性关系;并从迁移时间的基本方程定量地解释了上述的变化规律.本文还发现,采用相对迁移时间指标后可以有效地消除操作电流以及柱温对迁移时间所引起的波动,是毛细管区带电泳的一个特征参数.     

Nonaqueous capillary electrophoresis for the analysis of selected tropane alkaloids in a plant extract

Summary The potential of nonaqueous capillary electrophoresis has been investigated for the separation of structurally similar tropane alkaloids. The effects of the organic solvent and of electrolyte composition on separation selectivity, migration times, and efficiency are described. The addition of trifluoroacetic acid to the separation buffer was found beneficial for manipulation of the order of migration of the two positional isomers littorine and hyoscyamine. Replicate injections under nonaqueous conditions gave migration time and peak area data of excellent precision. The application of the optimized conditions to the analysis of hyoscyamine and scopolamine in genetically transformed root cultures ofDatura candida x D. aurea is presented.     

Determination of Seven Active Ingredients in Three Plant Essential Oils by Using Micellar Electrokinetic Chromatography

A simple micellar electrokinetic chromatography (MEKC) method was developed for determination of citronellal, citral (Z; E), α-pinene, limonene, linalool, and eugenol in plant essential oils (EOs). A buffer consisting of 20 mM Na₂B₄O₇, 50 mM SDS, 20% (v:v) methanol adjusted to pH 9.5 was found to provide a very efficient and stable electrophoretic system for the analysis. The validation of the method included linearity, LODs, LOQs, precision (intra - and inter - day variation of migration time and peak area), and recovery. Seven terpenoids presented good linearity (R ² > 0.9960) within the test ranges; LODs (S/N = 3) and LOQs (S/N = 10) were 0.2–1.8 µg/mL and 0.8–5.9 µg/mL, respectively. The precision and accuracy were satisfactory, with the overall intra- and inter-day variation (for migration time and peak area, RSD%) being less than 7.0%, and recoveries of this method were greater than 91% at spiked levels. The proposed method was successfully applied to the determination of seven terpenoids in clove oil, litsea cubeba oil, and citronella oil, respectively.     

Anionic microemulsion to solvent stacking for on‐line sample concentration of cationic analytes in capillary electrophoresis

The common SDS microemulsion (i.e. 3.3% SDS, 0.8% octane, and 6.6% butanol) and organic solvents were investigated for the stacking of cationic drugs in capillary zone electrophoresis using a low pH separation electrolyte. The sample was prepared in the acidic microemulsion and a high percentage of organic solvent was included in the electrolyte at anodic end of capillary. The stacking mechanism was similar to micelle to solvent stacking where the micelles were replaced by the microemulsion for the transport of analytes to the organic solvent rich boundary. This boundary is found between the microemulsion and anodic electrolyte. The effective electrophoretic mobility of the cations reversed from the direction of the anode in the microemulsion to the cathode in the boundary. Microemulsion to solvent stacking was successfully achieved with 40% ACN in the anodic electrolyte and hydrodynamic sample injection of 21 s at 1000 mbar (equivalent to 30% of the effective length). The sensitivity enhancement factors in terms of peak height and corrected peak area were 15 to 35 and 21 to 47, respectively. The linearity R² in terms of corrected peak area were >0.999. Interday precisions (%RSD, n = 6) were 3.3–4.0% for corrected peak area and 2.0–3.0% for migration time. Application to spiked real sample is also presented.     

Thermostating in capillary electrophoresis

Summary The use of high voltages across a electrophoresis capillary will increase the temperature of the buffer due to Joule heating. As a result temperature control in CE is rather important since variations in the buffer temperature will result in changes in the pH of the buffer, peak shape, migration time, reproducibility, efficiency, 3-D structure of macromolecular analytes, etc. Six different thermostating systems have been evaluated: (i) natural convection, (ii) fan, (iii) home-made and (iv and v) two commercially available high-speed air and a (vi) liquid thermostated device. In all cases the temperature of the buffer in the capillary is calculated according to the temperature-conductivity relationship. For this purpose two parameters are introduced describing temperature control: the temperature onset (δT) and the temperature rise factor (α). From these results, it can be concluded that high speed air thermostating can be as efficient as liquid thermostating.     

Determination of p-tyrosol and salidroside in three samples of Rhodiola crenulata and one of Rhodiola kirilowii by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method has been developed for simultaneous assay of two bioactive components (p-tyrosol and salidroside) in Rhodiola crenulata and Rhodiola kirilowii for the first time. Those analytes were successfully separated within 15 min using 50 mmol L^–1 (pH 9.62) borate containing 30% methanol as running buffer. Regression equations revealed linear relationships (correlation coefficients 0.9998–0.9999) between peak area and concentration of the two analytes. The relative standard deviations (RSD) of the migration times and the peak areas of two constituents ranged from 0.51 to 0.57% and from 0.65 to 1.17%, respectively, intra-day, and from 4.91 to 6.93% and from 3.51 to 5.33%, respectively, inter-day. The recoveries of two constituents ranged from 96.24 to 103.15%.     

Quantification of 5‐aminolevulinic acid by CE using dynamic pH junction technique

An online dynamic pH junction preconcentration method was developed for quantification of 5‐aminolevulinic acid (ALA) by CE with the separation time less than 6 min. The optimal dynamic pH junction of ALA was carried out between pH 9.3 borate buffer (BGE, 40 mM) and pH 2.5 phosphate buffer (sample matrix, 40 mM) when 4.1 cm of sample plug was hydrodynamically injected into an uncoated fused‐silica capillary (48.5 cm in length, id of 50 μm). If a 24 kV separation voltage was applied, the calibration curve of ALA peak area (200 nm) showed good linearity (R² = 0.9991) ranging from 0.01 to 6.5 mg/mL. The reproducibility of this system was excellent with RSDs (n = 10) of 2.5% for peak area response and 0.6% for migration time at ALA concentration of 0.5 mg/mL. The LOD was evaluated as 1.0 μg/mL (S/N > 3). Compared to conventional CE procedure, the sensitivity was successfully improved over 50‐fold. The analytical results of pharmaceutical formulations show a good agreement with those by HPLC (r = 0.94).     

Expanding the scope of pressure‐assisted electrokinetic injection for online concentration of positively charged analytes in capillary electrophoresis‐mass spectrometry

Sample injection is a crucial step in CE. In past, many efforts have been focused on concentrating the analytes into a sharp sample plug. In 2006, pressure‐assisted electrokinetic injection (PAEKI) was proposed as a new preconcentration technique for anions. In this study, we expanded the applicability of PAEKI to online preconcentrate positively charged analytes. l ‐Arginine, l ‐lysine, and imidazole were chosen as target analytes for method development. The enhancement factor of PAEKI was over 3000‐fold. When CZE was coupled with a Q‐TOF system, PAEKI delivers a detection limit of 18–28 pg/mL and a dynamic calibration range over four orders of magnitude. The RSD was less than 6.4% (n = 4) on both peak area and migration time, indicating a good repeatability.     

Electropherograms in capillary zone electrophoresis plotted as a function of the quantity of electric charge

We have plotted electropherograms in capillary zone electrophoresis (CZE) as a function of the quantity of electric charge (Q) in order to eliminate the dependency of the analyte peak areas, as well as that of the migration times, upon both the capillary temperature and the applied voltage. The procedure is based on an idea of a migration index (MI) and an adjusted migration index (AMI) which were originally proposed by Lee and Yeung. The value of Q is measured accurately and calculated easily because it is given by a product of the electrophoretic current and the migration times, where the index MI is derived by dividing the value of Q by the effective volume of the capillary. By calculating the CZE peak area from the newly plotted electropherogram, improvement in precision in quantitative analysis is expected. Concerning AMI, careful treatment is required in its application to analyte peaks whose migration time is close to that of the neutral marker. Experimental data and discussions concerning the migration indices are presented.     

Simultaneous Determination of the Active Ingredients in Abelmoschus manihot (L.) Medicus by CZE

A capillary zone electrophoretic method has been developed for the quantitative analysis of four active compounds, quercetin-3-O-robinobioside (LXY3), hyperin (LXY4), isoquercetin (LXY5) and myricetin (LXY7) in A. manihot (L.) Medicus with UV detection at 254 nm. The capillary temperature was kept constant at 25 °C. The effects of buffer composition, pH, and concentration and running voltage on migration were studied systematically. Optimum separation was achieved with 20 mmol L^?1 Borax–NaOH buffer (pH 9.50) at 25 °C and 25 kV. Regression equations revealed good linear relationship between the peak area of each compound and its concentration. All the correlation coefficients were higher than 0.9990. The relative standard deviations of migration time and the peak area were <0.53% and 5.34% (inter-day), and <0.56% and 3.81% (intra-day). The contents of the four compounds in A. manihot (L.) Medicus were successfully determined with satisfactory repeatability and recovery.     

Highly efficient high‐performance liquid chromatographic separation of xylene isomers and phthalate acid esters on a homemade DUT‐67(Zr) packed column

In this study, the baseline separations of xylene isomers and phthalate acid esters on a homemade DUT‐67(Zr) packed column were achieved, respectively. The high selectivity for xylene isomers and phthalate acid esters was obtained with the increase in temperature and decrease in the retention time. The hydrophobicity of xylene isomers and phthalate acid esters resulted in the different separation time on the DUT‐67(Zr) packed column. The relative standard deviation values of retention time, peak area, peak height, and half peak width for five repeat separation of the xylene isomers were 0.26–0.35, 2.11–2.26, 1.51–2.03, and 0.29–0.77%, and the values of the phthalate acid esters on DUT‐67(Zr) column were 0.1–0.4, 4.4–5.2, 3.9–6.3, and 0.6–2.1%, respectively. The thermodynamic properties indicated that the separation of xylene isomers was controlled by ΔH and ΔS, but the separation of phthalate acid esters was mainly controlled by ΔS.     

Separation and determination of honokiol and magnolol in herbal medicines by flow injection-capillary electrophoresis

A simple, rapid, and accurate method for the separation and determination of honokiol and magnolol in Magnolia officinalis and related herbal medicines was developed by combination of flow injection (FI) and capillary zone electrophoresis (CZE). The analysis was carried out using an unmodified fused-silica capillary (50-μm I.D.; total length 7.5 cm; effective length 4.5 cm). A series of optimization steps afforded the following conditions: the sample solvent consisted of 150 mM NaOH and a running buffer composed of 10 mM sodium tetraborate/10 mM sodium dihydrogenphosphate (NaH₂PO₄) at pH 12 was applied for the separation of the analytes. The separation could be achieved within 5 min with a sample throughput rate of up to 28 h⁻¹. The repeatability (defined as the relative standard deviation, RSD) for honokiol and magnolol was 2.0% and 1.6% with peak area evaluation, 3.6% and 2.0% with peak height evaluation, and 2.0% and 1.4% with migration time evaluation, respectively. Regression equations revealed linear relationships (r = 0.9991–0.9998) between the peak area of each analyte and the concentration.     

Precision of micellar electrokinetic capillary chromatography in the determination of seven antibiotics in pharmaceuticals and feedstuffs

Validation of analytical procedures is important for their efficient and reliable application. The International Conference on Harmonisation (ICH), Food and Drug Administration (FDA) and pharmacopoeia guidelines achieved a great deal in harmonising the definitions of the required validation characteristics. It is well known that poor reproducibility limits the practical implementation of capillary electrophoresis (CE). A precision study on four different MEKC methods was performed with 11 samples, containing seven antibiotics, by two analysts, in few days, on two capillary electrophoresis instruments. Five pharmaceutical preparations and three animal feeds were used. Precision was statistically analysed using migration time, peak area and height of each compound, as well as electroosmotic front (EOF). In 25 of 31 cases, the reproducibility of peak area, peak height and migration time was good (<5%). In most cases the reproducibility of peak area was much better than the reproducibility of peak height. The worst reproducibility that we observed was 12.7% for peak height and 7.6% for peak area.     

Mobile phase additives for enhancing the chromatographic performance of astaxanthin on nonendcapped polymeric C30‐bonded stationary phases

Astaxanthin shows peak deformation and reduced peak area response when eluted with methanol and methyl tert‐butyl ether on nonendcapped polymeric C₃₀‐bonded HPLC phases. The present study tested different column manufacturers, column batches, and ten mobile phase additives including acids, bases, buffers, complexing and antioxidant agents for improvement of peak shape and peak area response. Concerning chromatographic benefits and feasibility, ammonium acetate was found to be the best additive followed by triethylamine for all columns tested. Variation of the mobile phase pH equivalent and the column temperature showed no synergistic effects on peak shape and peak area response. Results indicate that peak tailing and variation of peak area response are due to different on‐column effects. Possible mechanisms of the observed phenomenon will be discussed.