Analysis of large-volume DNA markers and polymerase chain reaction products by capillary electrophoresis in the presence of electroosmotic flow

We have demonstrated on-line concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large-volumes DNA samples, PEO solutions entered a capillary filled with 400 mM Tris-borate (TB) buffers by EOF and acted as sieving matrices. DNA fragments stacked between the sample zone and PEO solutions. Because sample matrixes affected PEO adsorption on the capillary wall, leading to changes in EOF, migration time, concentration, and resolving power varied with the injection length. When injecting phiX174 RF DNA-HaeIII digest prepared in 5 mM Tris-HCl buffer, pH 7.0, at 250 V/cm, peak height increased linearly as a function of injection volume up to 0.9 microl (injection time 150 s). The sensitivity improvement was 100-fold compare to that injected at 25 V/cm for 10 s (0.006 microl). When injecting 1.54 microl of GeneScan 1000 ROX, the sensitivity improvement was 265-fold. The sensitivity improvement was 40-fold when injecting 0.17 microl DNA sample containing pBR 322/HaeIII, pBR 328/BglI, and pBR 328/HinfI digests prepared in phosphate-buffered saline. This method allows the analysis of polymerase chain reaction (PCR) products amplified after 17 cycles when injecting 0.32 microl (at 30 cm height for 300 s). The total analysis time was shorter (91.6 min) than that (119.6 min) obtained from injecting PCR products after 32 cycles for 10 s.     

Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.     

Stepwise capillary electrophoretic separation of DNA fragments using poly(ethylene oxide) solutions in the presence of electroosmotic flow.

Single-base resolution in the separation of DNA markers V and VI was achieved in the presence of electroosmotic flow (EOF), using poly(ethylene oxide) (PEO) solutions containing ethidium bromide (EtB) under isocratic conditions. Furthermore, a new approach called stepwise capillary electrophoresis (SCE) has been developed for DNA analysis, including stepwise changes in PEO concentration, EtB concentration as well as both PEO and EtB concentrations, wherein the EOF was used to introduce different PEO solutions into the capillary during the separation. DNA fragments smaller than 80 bp were both detected under isocratic conditions using 20 micrograms/ml EtB, and SCE using 1 and 20 micrograms/ml EtB, but not under isocratic conditions using 1 microgram/ml EtB. Resolution and speed of the DNA separation in SCE were different from those obtained from isocratic means, indicating that DNA underwent different concentrations of PEO and EtB in SCE. For example, DNA fragments with 458 and 504 base pairs (bp) were partially resolved in SCE, but not under isocratic conditions. The results further suggest that it is worth developing gradient techniques for widening the separation range and enhancing resolution in DNA analysis.     

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.     

On-column preconcentration and separation of DNA fragments using polymer solutions in the presence of electroosmotic flow

We demonstrated DNA preconcentration and separation in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large volumes of DNA samples into a capillary filled with free tris(hydroxymethyl)aminomethane (Tris)-borate (TB) buffers, PEO solutions entered the capillary by EOF and acted as sieving matrices. In contrast to conventional methods (in the absence of EOF), controlling the EOF was also useful for resolution optimization. We have found that PEO adsorption on the capillary wall was more pronounced when low ionic strength buffers were used. Thus, the EOF decreased with increasing injection length, which led to longer migration times and changes in resolution and stacking efficiency. All resolution values were higher than 1.5 when 1.0 microg/mL DNA samples were injected at 240 V/cm for 60 s (0.67 microL). In addition, as low as 0.015 microg/mL DNA samples (an about 66-fold increase in sensitivity) were detected when the injection was performed at 250 V/cm for 60 s.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.     

A new strategy for optimizing sensitivity, speed, and resolution in capillary electrophoretic separation of DNA

DNA separations were performed in poly(ethylene oxide) (PEO) solutions prepared in 100 mM Tris-boric acid (TB) buffers using a capillary filled with TB buffers with concentrations up to 2.5 M, pH 10.0. The electroosmotic flow (EOF) increased with increasing the concentration of TB buffers till 1.5 M as a result of decreasing PEO adsorption on the capillary wall. At high TB concentrations (> 1.5 M), the peaks corresponding to small DNA fragments (11 and 8 base pairs) became sharper and were detected. Relative standard deviations of the EOF coefficient and the migration times of the DNA fragments were all less than 1% using a capillary filled with TB buffers at concentrations higher than 1.5 M. When separations were performed at different pH values of PEO solutions and TB buffers, better results in terms of sensitivity, speed, and resolution were generally achieved. The fluorescence intensity of the 2176 bp fragment obtained at pH values of TB buffers/PEO solutions 10.0/8.2 was 27-fold of that at pH values 8.2/8.2. The enhancement was related to effects of pH and borate on fluorescence intensity, DNA conformation, stacking, and interactions with the capillary wall. Using a capillary filled with 400 mM TB buffers, pH 10.0, the separation of DNA (pBR 322/HaeIII digest, pBR 328/Bg/I digest and pBR 328/HinfI digest) in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.     

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

The impact of a plug of salts on the analysis of large volumes of dsDNA by capillary electrophoresis

A partially filling technique for the analysis of DNA markers and polymerase chain reaction (PCR) products by capillary electrophoresis in the presence of electroosmotic flow using polymer solutions is presented. Either after or prior to the sample injection, a plug of salts at high pH was hydrodynamically injected. During the separation, poly(ethylene oxide) (PEO) solution entered the capillary. We have found that the position, length, and composition of the plugs affect the sensitivity, resolution, and speed on the analysis of PhiX-174/HaeIII DNA restriction fragments or a DNA mixture (pBR 322/HaeIII digest, pBR 328/BglI digest and pBR 328/HinfI digest) with different degrees. Through careful evaluation of the impact of anions and cations on the analysis of DNA, we have suggested that the optimal condition is applying a plug consisting of 32 mM NaCl and 0.01 M NaOH at 30 cm height for 60 s after sample injection. In the presence of such a plug, PEO adsorption reduces, and thus the separation is faster, as well as the sensitivity improves. Using this condition, the analysis of a DNA mixture (injected at 30 cm for 360 s) containing ten different PCR products amplified after 17 cycles was complete in 25 min. About a 2000-fold improvement in the sensitivity was achieved when compared to that by a conventional method (10 s injection) without applying a plug.     

Analysis of double-stranded DNA by capillary electrophoresis using poly(ethylene oxide) in the presence of hexadecyltrimethylammonium bromide

The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by capillary electrophoresis in conjunction with laser-induced fluorescence (CE-LIF) detection using poly(ethylene oxide) (PEO) solution is described. The use of CTAB for improved separation reproducibility and efficiency of DNA has not been demonstrated although it is widely used for controlling the magnitude and direction of electroosmotic flow in CE. With increasing CTAB concentration, the interactions of DNA with ethidium bromide (EtBr) and with the capillary wall decrease. For the separation of DNA fragments with the sizes ranging from several base pairs (bp) to 2,176 bp, a polymer solution consisting of 0.75% poly(ethylene oxide), 100 mM TB buffer (pH 8.0), 25 microg/mL EtBr, and 0.36 microg/mL CTAB is proper. Using the PEO solution, we separated a mixture of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BglI digest and pBR 328/HinfI digest) within 8 min at -375 V/cm, with the limit of detection of 2.0 ng/mL based on the peak height for the 18-bp DNA fragment. The method is highly efficient (>10(6)plate/m), repeatable (RSD of the migration times <1.5%), and sensitive. In addition, it is convenient to fill a capillary (75 microm in diameter) with such a low-viscosity PEO solution by syringe pushing.     

Separation of double-stranded DNA fragments by capillary electrophoresis: Impacts of poly(ethylene oxide), gold nanoparticles, ethidium bromide, and pH

The separation of DNA by capillary electrophoresis using poly(ethylene oxide) (PEO) containing gold nanoparticles (GNPs) is presented. The impacts of PEO, GNPs, ethidium bromide (EtBr), and pH on the separation of double-stranded DNA have been carefully explored. Using a capillary dynamically coated with 5.0% poly(vinylpyrrolidone) and filled with 0.2% PEO containing 0.3 x GNPs (the viscosity less than 15 cP), we have demonstrated the separation of DNA markers V and VI within 5 min at pH 8.0 and 9.0. In terms of resolution and reproducibility, GNPs have a greater impact on the separation of DNA at pH 9.0. Resolution improvements for large DNA fragments (> 300 base pairs, bp) are greater than those for small ones in the presence of GNPs. It is important to point out that reproducibility is excellent (relative standard deviations for the migration times less than 0.5%) and thus no further dynamic coating is required in at least 20 consecutive runs in the presence of GNPs. Using 0.2% PEO (pH 9.0) containing 0.3 x GNPs, the separation of DNA fragments ranging in size from 21 to 23,130 bp was accomplished in 7 min. The results presented in this study show the advantage of PEO containing GNPs for DNA separation, including rapidity, high resolving power, excellent reproducibility, and ease of filling capillaries.     

On-line pre-concentration and UV determination of DNA fragments by dynamic coating capillary electrophoresis and its application to detection of genetically modified oilseed rape based on PCR

In this work, it was demonstrated that on-line pre-concentration and separation of DNA fragments within bared silica column by dynamic coating capillary electrophoresis and UV detection. The DNA fragments were pre-concentrated with long electrokinetic injecting time (99 s), peak height increased dramatically as a function of injection time, especially for shorter length DNA. The concentration sensitivity of DNA fragments can be improved from 20- to 100-fold relative to a normal injection (5 s). The electro-osmotic flow (EOF) and DNA-wall interactions within the capillary were eliminated effectively by dynamic coating method. Employing 0.5% poly(ethylene oxide) (PEO) in Tris-phosphate-EDTA (TBE) buffer as sieving matrix, DNA fragments, ranging from 11 to 657 bp, were separated within 20 min. The linear coefficient of linear relation between the migration and DNA length is 0.999. The DNA fragments amplified from transgenic oilseed rape by polymerase chain reaction (PCR) were separated and detected by this method, demonstrating the potential use of this method for effective DNA analysis and detection of genetically modified organisms (GMO).     

Fast and sensitive diagnosis of thalassemia by capillary electrophoresis

This paper demonstrates the diagnosis of -thalassemia by capillary electrophoresis in conjunction with laser-induced fluorescence using poly(ethylene oxide) (PEO) solutions in the presence of electroosmotic flow (EOF). During the electrophoretic separation, PEO solution entered a capillary from the anodic vial by EOF. The separation of a mixture of the polymerase chain reaction (PCR) products (330 and 334 base pairs) from a healthy person and a -thalassemia patient was accomplished within 15 min at 15 kV using 1.5% PEO containing 2 M urea at 30 °C. The electropherogram patterns instead of migration times were used to diagnose -thalassemia, with an accuracy of 100% for the analyses of 11 blood samples from suspected patients. After injecting a large volume of the mixture to the capillary filled with 800 mM Tris-borate buffer (pH 10.0), the DNA fragments stacked due to increases in viscosity and sieving when migrating into 1.5% PEO solution. As a result of improved sensitivity, only 15 PCR cycles were required when using 500 ng of DNA templates. The results shown in this study indicate the potential of this simple, rapid, and cost-effective method for the diagnosis of -thalassemia.Abbreviations CE Capillary electrophoresis - EOF Electroosmotic flow - EtBr Ethidium bromide - LIF Laser-induced fluorescence - PCR Polymerase chain reaction - PEO Poly(ethylene oxide) - TRIS Tris(hydroxymethyl)aminomethane - TB TRIS-borate     

Improved separation of double-stranded DNA fragments by capillary electrophoresis using poly(ethylene oxide) solution containing colloids

The analysis of double-stranded (ds) DNA fragments by capillary electrophoresis (CE) using poly(ethylene oxide) (PEO) solution containing gold nanoparticles (GNPs) is presented, focusing on evaluating size dependence of the GNPs and PEO on resolution and speed. To prevent the interaction of the capillary wall with DNA, the capillary was dynamically coated with polyvinylpyrrolidone. Using different PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm, we have achieved reproducible, rapid, and high-resolution DNA separations. The results indicate that the sizes of PEO and GNPs as well as the concentration of PEO affect resolution. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO (8 MDa) containing 56 nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO (2 MDa) containing 13 nm GNPs or 0.05% PEO (4 MDa) containing 32 nm GNPs. With very low viscosity (< 15 cP), automatic replacement of the sieving matrices is easy, indicating a great potential for high-throughput DNA analysis using capillary array electrophoresis systems.     

Capillary electrophoretic separation of dsDNA under nonuniform electric fields

Improved sensitivity for the analysis of DNA by capillary electrophoresis has been achieved, based on simultaneous increases in optical path length and injection volume. To increase the optical path length, bubble cells with diameters ranging from 150 to 450 microm have been fabricated and tested. In terms of resolution and sensitivity, a bubble cell of 300 microm diameter is appropriate when using 75-microm capillaries. To allow greater injection volumes, we performed on-line concentration of DNA in the presence of electroosmotic flow (EOF) using 2.0% poly(ethylene oxide) (PEO). With a 300-microm bubble cell, a 170-fold improvement in the sensitivity for the 89-bp fragment has been accomplished when injecting about 0.33 microL DNA. In the presence of the bubble cell, the resolution for the large fragments improves while that for the small ones (<124 base pair) decreases. The effect of bubble cells was further investigated by conducting DNA separation in the absence of EOF, showing that improvements in resolution are mainly due to increased migration differences when DNA migrated at low electric field strengths in the bubble region. We have suggested that such an effect is more profound using shorter capillaries, leading to complete separation of phiX 174 RF DNA-Hae III digest in 2 min.     

Stacking, derivatization, and separation by capillary electrophoresis of amino acids from cerebrospinal fluids

This paper describes the in-column derivatization, stacking, and separation of amino acids by CE in conjunction with light-emitting diode-induced fluorescence using naphthalene-2,3-dicarboxaldehyde (NDA). According to the relative electrophoretic mobilities and the migration direction in tetraborate solution (pH 9.3), the injection order is cyanide, then amino acids, then NDA. Once poly(ethylene oxide) (PEO) migrates through the capillary under EOF, the amino acid.NDA derivatives, amino acids, and CN- ions migrating against the EOF enter the PEO zone. As a result of increases in viscosity and possible interactions with PEO molecules, the reagents/analytes slow down such that they become stacked at the boundary. In comparison with the off-column approach to the analysis of amino acids, our proposed method provides a lower degree of interference from polymeric NDA compounds and other side products. As a result, the plot of the peak height as a function of gamma-aminobutyric acid (GABA) concentration is linear over the range from 10(-5) to 10(-8) M, with the LOD being 4 nM. We demonstrate the diagnostic potential of this approach for the determination of amino acids, including GABA and glutamine, in biological samples through the analysis of large volumes of cerebral spinal fluids without the need for sample pretreatment.     

High-resolution DNA separation in microcapillary electrophoresis chips utilizing double-L injection techniques

An experimental and numerical investigation into the use of high-resolution injection techniques to separate DNA fragments within electrophoresis microchips is presented. The principal material transport mechanisms of electrokinetic migration, fluid flow, and diffusion are considered, and several variable-volume injection methods are discussed. A detailed analysis is provided of a double-L injection technique, which employs appropriate electrokinetic manipulations to reduce sample leakage within the microchip. The leakage effect in electroosmotic flow (EOF) is investigated using a sample composed of rhodamine B and Cy3 dye. Meanwhile, the effects of sample leakage in capillary electrophoresis (CE) separation are studied by considering the separation of 100-base pairs (bp) DNA ladders and HaeIII-digested PhiX-174 DNA samples. The present experimental and simulation results indicate that the unique injection system employed in the current microfluidic chip has the ability to replicate the functions of both the conventional cross-channel and the shift-channel injection systems. Furthermore, applying the double-L injection method to these two injection systems is shown to reduce sample leakage significantly. The proposed microfluidic chip and double-L injection technique developed in this study have an exciting potential for use in high-resolution, high-throughput biochemical analysis applications and in many other applications throughout the micrototal analysis systems field.     

单链构象多态性-毛细管电泳分析中样品纯化研究

聚合酶链反应(PCR)样品中的Cl^-、引物等小分子对单链构象多态性一毛细管电泳分析有重要影响。本文用毛细管电泳一间接紫外法测定了PCR样品中的Cl^-浓度,并对乙醇沉淀法和试剂盒纯化法用于PCR样品的纯化效果进行比较,在此基础上研究了Cl^-浓度、引物及其二聚体在SSCP-CE分析中的影响。结果表明,它们主要影响DNA进样量而对分离效率影响不大。     

Constant pressure-assisted electrokinetic injection for on-line enhanced detection of monophthalates in capillary electrophoresis-mass spectrometry with application to human urine

Electrophoresis characteristics of several monophthalates in the sample zone and EOF variation in a fused-silica capillary column during a constant pressure-assisted electrokinetic injection (PAEKI) in an on-line CE-MS were studied in an effort to reconcile the mobility theory and field amplification with the enhancement achieved in present work. Influences of capillary length on the amount injected using PAEKI were investigated in detail and except for the injection time, the amount injected was found to increase linearly with capillary column length. A longer capillary provides a longer linear increase time range with PAEKI injection. The results show that smaller m/z analytes generate a large enhancement power using PAEKI, which is in agreement with the mobility theory. ACN was used as an example to investigate influences of organic additives on the amount injected and was found to decrease the amount injected with PAEKI injection, which is in agreement with an increase of resistivity in running buffer by organic additives. The peak width obtained with PAEKI injection proved to be independent of the amount injected. The band size of the sample zone was estimated by comparison with conventional hydrodynamic injection. A 240 s PAEKI injection achieved the same size of sample zone as a 2 s of hydrodynamic injection. Existance of two ion layers around the boundary of the buffer and sample solutions in sample zone was hypothesized to contribute the narrow sample zone with a long time of PAEKI injection. With a 240 s on-line PAEKI injection in CZE-MS, five monophthalates were enriched several hundred times without compromise in their separation efficiency and peak shape. With appropriate sample cleanup, PAEKI was applied to the analysis of monophthalates in urine samples, achieving detection limits ranging between 0.53 and 1.3 ng/mL.     

Sample stacking by field-amplified sample injection and sweeping for simultaneous analysis of acidic and basic components in clinic application

In this study, online sample concentration method, which coupled field-amplified sample injection (FASI) and sweeping technology with micellar electrokinetic chromatography (MEKC), was used to detect and analyze acidic and basic components in a single run. In order to concentrate the acidic and basic components simultaneously in a single run sweeping step, a combination of successive anion- and cation-selective injections were used. Before sample loading, a rinse buffer containing 50 mM Tris buffer (pH 3) with 41% MeOH and 0.1% polyethylene oxide (PEO) was injected in order to suppress the electroosmotic flow (EOF). Sample loading of anionic components was achieved by electrokinetic injection at a negative voltage of -2.5 kV for 80 s, and then the cationic components were injected at a positive voltage of +5 kV for 120 s. Finally, sweeping with SDS micelles from the separation buffer (25 mM Tris buffer with 60 mM SDS, pH 3) was performed at a negative voltage of -20 kV. This capillary electrophoretic methodology was applied to the quantification of acidic and basic drugs in commercial tablets and in plasma samples. The precision and accuracy of the proposed method at different concentrations ranging from high, medium, to low were evaluated on spiked plasma samples. The intra and interday precision and accuracy values at three concentrations were all below 6.1%. The method was also successfully applied to monitor the tested drugs in the plasma of nine elderly cardiovascular and/or Alzheimer's disease patients after oral administration of the commercial products.