Development and validation of HPLC method for the determination of α-tocopherol in human erythrocytes for clinical applications

In this work, a simple isocratic reversed-phase HPLC method for determination of alpha-tocopherol in human erythrocytes has been developed and validated. After separation of plasma the erythrocytes were washed three times with 0.9% sodium chloride containing 0.01% butylated hydroxytoluene (BHT) as antioxidant and then were diluted 1:1 (v/v) with the same solution. In the liquid-liquid extraction (LLE) procedure, 2500 microL of n-hexane was added to 500 microL of erythrocytes. After 2 min this mixture was deproteinized by addition of cool ethanol (500 microL, 5 min) denatured with 5% methanol containing alpha-tocopherol acetate (20 micromol L(-1)), as internal standard, and then extracted for 5 min by vortex mixing. After centrifugation (10 min, 1600xg) an aliquot (2000 microL) of the clean extract was separated and evaporated under nitrogen. The residue was dissolved in 400 microL methanol and analysed by reversed-phase HPLC on a 4.6 mmx150 mm, 5 microm Pecosphere C18 column; the mobile phase was 100% methanol, flow rate 1.2 mL min(-1). The volume injected was 100 microL and detection was by diode-array detector at a wavelength of 295 nm. The extraction recovery of alpha-tocopherol from human erythrocytes was 100.0+/-2.0%. The detection limit was 0.1 micromol L(-1) and a linear calibration plot was obtained in the concentration range 0.5-20.0 micromol L(-1). Within determination precision was 5.2% RSD (n=10), between determination precision was 6.1% RSD (n=10). The method was applied successfully in a clinical study of patients with acute pancreatitis and for determination of the reference values in the healthy Czech population.     

A new strategy for supercritical fluid extraction of copper ions

Complexation combined with supercritical fluid extraction was used to extract Cu²⁺. The effects of pressure, temperature, and total volume of CO₂ on the efficiency of extraction were systematically investigated. The extraction recovery was low (57.32%) only by pure supercritical CO₂. Addition of a suitable amount of methanol (v/v=5%) to supercritical CO₂ could enhance the extraction of Cu²⁺ (72.69%, relative standard deviation (R.S.D.)=2.12%, n=3), and the recovery increased largely (90.52%, R.S.D.=2.20%, n=3) in the presence of nonionic surfactant Triton X-100. Reverse micelle formation is presented as a new strategy of improving the extraction of metal ions with supercritical CO₂ in this paper.     

A rapid cleanup method for the isolation and concentration of pyrrolizidine alkaloids in comfrey root

Preparations from comfrey (Symphytum officinale and S. x uplandicum) root and leaf contain varying levels of the hepatotoxic pyrrolizidine alkaloids (PAs). Reference compounds for comfrey are not commercially available, and there is currently no rapid extraction or analytical method capable of determining low levels in raw materials or as adulterants in commercially available extracts. A solid-phase extraction (SPE) method was developed using an Ergosil cleanup column that specifically binds the PAs. With this method, powdered comfrey root was extracted by sonication and shaking with basic chloroform. The extract was applied to the cleanup column under vacuum, washed with 2 mL acetone-chloroform (8 + 2, v/v) followed by 2 mL petroleum ether to remove excess chloroform. The column was dried under vacuum, and the PAs were eluted with 2 successive 1 mL aliquots methanol. Percent recoveries of the PAs following Ergosil SPE had an overall average of 96.8%, with RSD of 3.8% over a range of 1.0 to 25.0 g extracted in 100 mL. Average precision of the method (n = 3 over 4 extraction concentrations) gave an overall RSD of 6.0% for the 5 alkaloids, with a range of 0.8% (5 g in 100 mL) to 11.2% (25 g in 100 mL). Recovery optimization testing showed that 1.0 g comfrey root extracted in 100 mL yielded the greatest recovery (% dry weight) of the PAs, with an extraction efficiency and accuracy of 94.2%, and RSD of 1.7% (n = 9). The unique properties of the Ergosil cleanup column provide rapid sample cleanup, volume reduction, and concentration of PAs from comfrey extracts, and allow the eluant to be analyzed directly by traditional chromatographic methods.     

Supercritical carbon dioxide extraction of ubiquinones and menaquinones from activated sludge

Supercritical CO2 (scCO2) extraction, with methanol as modifier, was applied to the determination of ubiquinones and menaquinones in activated sludge. Four ubiquinones and 12 menaquinones species were identified based on retention time and UV spectrum in 0.1g dried activated sludge. The optimum extraction conditions were at a pressure of 25 MPa, a temperature of 55 degrees C, and 10% (v/v) methanol for 15 min. At this condition, the concentrations of extracted ubiquinones and menaquinones were found to be 0.181 and 0.326 micromol/g-dry-cell, respectively. The results were comparable with those obtained by organic solvent extraction based on diversity and dissimilarity indices. Furthermore, the method was evaluated in term of repeatability, which resulted in an RSD of < or =10%. The experimental results have demonstrated the technique to be simple, fast, and with less consumption of organic solvents. This work shows the potential application of supercritical CO2 extraction to microbial community analysis using quinone profile.     

Effects of different parameters on supercritical fluid extraction of steroid drugs, from spiked matrices and tablets

Feasibility of supercritical CO(2) extractions of two steroid drugs, medroxyprogesterone acetate (med) and cyproterone acetate (cyp), were evaluated. The effects of temperature (308-348 K), pressure (100-300 bar), static extraction time (5-15 min), dynamic extraction time (10-30 min) and percent methanol modifier (1-10% v/v) on the SFE recoveries of these drugs from spiked matrices (glass) and pharmaceutical dosages (tablets) were investigated. The results showed that minor structural differences between related compounds might lead to dramatically differences in extraction behaviors under the same conditions. The optimum SFE conditions to extract the drugs from spiked glass were 10 min static, 30 min dynamic, 300 bar, 348 K and 5% modifier in the case of med acetate and 10 min static, 30 min dynamic, 100 bar, 308 K, and 10% modifier in the case of cyp. Under these conditions above 90% of the total recovery was obtained for both drugs. Extractions from pharmaceutical dosages were less efficient compared to glass beads under the same conditions. Quantitative recovery of solutes from tablets were obtained upon changing extraction conditions to: 15 min static, 45 min dynamic, P=300 bar, 20% modifier (for med) and 10% (for cyp).     

Supercritical fluid extraction and gas chromatography analysis of arsenic species from solid matrices

A method in combination with derivatization-supercritical fluid extraction(SFE) and gas chromatography(GC) for the speciation and quantitative determination of dimethylarsinate(DMA), monomethylarsonate(MMA) and inorganic arsenic in solid matrices was investigated. Thioglycolic acid methyl ester(TGM) and thioglycolic acid ethyl ester(TGE) were evaluated as derivatization reagents. The effects of pressure, temperature, flow rate of supercritical CO_2, extraction time, modifier and microemulsion on the efficiency of extraction were systematically investigated. The procedure was applied to the analysis of real soil and sediment samples. Results showed that TGE was more effective for arsenic speciation as a derivatization reagent. Modifying supercritical CO_2 with methanol can greatly improve the extraction efficiency. Further, the addition of microemulsion containing surfactant Triton X-100 can further enhance recoveries of arsenic species. The optimum extraction conditions were 100 ℃, 30 MPa, 10 min static and 25 min dynamic extraction with 5%(v/v) methanol, and surfactant modified supercritical CO_2. Detection limits in solid matrices were 0.15, 0.3 and 1.2 mg/kg for DMA, MMA and inorganic arsenic,respectively. The method was validated by the recovery data. The resulting method was fast, easy to perform and selective in the extraction and detection of various arsenic species in solid matrices.     

Interlaboratory verified liquid chromatographic method for analysis of vitamins A and E in soy-based infant formula powder

An interlaboratory verified, liquid chromatographic (LC) method is presented for the analysis of all-rac-alpha-tocopheryl acetate and retinyl palmitate in soy-based infant formula. The extraction procedure uses sample dehydration with magnesium sulfate followed by extraction with isopropanol, hexane-ethyl acetate (85 + 15, v/v). After evaporation and filtration, the sample extract is injected directly onto a normal-phase LC system with fluorescence detection. All-rac-alpha-tocopheryl acetate and retinyl palmitate are quantitated isocratically with a mobile phase of hexane containing 0.50% (v/v) and 0.125% (v/v) isopropanol, respectively. A zero control reference material (ZRM) was spiked at 5 levels, with 5 replicate analyses of 1/2x, x, 2x, 4x, and 16x where "x" is the minimum level of 250 IU/100 kcal (vitamin A) and 0.7 IU/100 kcal (vitamin E) as specified in 21 Code of Federal Regulations 107.100. The following recoveries and RSD values represent an average (n = 25) of the 5 levels for each analyte: all-rac-alpha-tocopheryl acetate, 100% (RSD = 3.5%); retinyl palmitate, 97.2% (RSD = 2.1%). Two additional laboratories analyzed the fortified ZRM samples. Average recoveries (n = 24) of all-rac-alpha-tocopheryl acetate and retinyl palmitate at 4 levels were all-rac-alpha-tocopheryl acetate, 99.0% (RSD = 4.0%), and retinyl palmitate, 96.2% (RSD = 1.4%) at the second laboratory. Average recoveries (n = 24) of all-rac-alpha-tocopheryl acetate and retinyl palmitate at 4 levels were all-rac-alpha-tocopheryl acetate, 102% (RSD = 1.4%) and retinyl palmitate, 95.7% (RSD = 2.0%) at the third laboratory. In addition, 6 replicates of the same commercial soy-based infant formula powder were run by the 3 laboratories.     

Supercritical fluid extraction of aurentiamide acetate from Patrinia villosa Juss and subsequent isolation by silica gel and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of aurentiamide acetate from Patrinia villosa Juss was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction (SFE) system. Then the extraction was scaled up by 100 times using a preparative SFE system under the optimized conditions of 55 degrees C, 35 MPa and modified CO2 with 10% methanol. Then, the crude extract I obtained by SFE was chromatographed on silica gel and the solvent system composed of petroleum ether-ethyl acetate (5:1, v/v) was used to produce the crude extract II, which was further isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:1.2:1.2:1, v/v/v/v). One hundred fifty-five milligrams of aurentiamide acetate was obtained from 400 mg crude extract II (contained 42% target) with a purity of 99.3% determined by HPLC and 92.3% recovery in one-step elution, and identification was performed by UV, MS, 1H NMR and 13C NMR. As far as we know, this is the first report of discovering aurentiamide acetate from the plant of Patrinia genius.     

Supercritical fluid extraction of organic and inorganic mercury from solid materials

Mercuric ions (Hg(2+)) can be extracted from solid samples (cellulose matrix) using methanol modified supercritical CO(2) containing the fluorinated chelating agent lithium bis(trifluoroethyl)dithiocarbamate (LiFDDC). Methylmercuric chloride (CH(3)HgCl) and dimethylmercury [(CH(3))(2)Hg] can be extracted by supercritical CO(2) without chelating agent and modifier. The solubility of Hg(FDDC)(2) in supercritical CO(2) has been determined to be 5 x 10(-3)M at 5O degrees C and 150 atm, which is about 3 orders of magnitude greater than that of the non-fluorinated analogue Hg(DDC)(2). Use of methanol (5%)-modified CO(2) further enhances the solubility of Hg(FDDC)(2) by a factor of 2.4. A small amount of water added to the sample matrix tends to facilitate the extraction of Hg(FDDC)(2) and CH(3)HgCl. Potential applications of this in situ chelation-supercritical fluid extraction method for the preconcentration of mercury species and treatment of mercury contaminated wastes are discussed.     

Quantitative determination of clozapine in serum by instrumental planar chromatography

An instrumental planar chromatographic (HPTLC) method for quantitative analysis of clozapine in human serum was developed and validated. Clozapine was extracted with n-hexane-isoamyl alcohol (75:25 v/v). The chromatographic separation was achieved on precoated silica gel F 254 HPTLC plates using a mixture of chloroform and methanol (9:1 v/v) as mobile phase. Quantitative analyses were carried out by densitometry at a wavelength of 290 nm. The method was linear between 10 and 100 ng/spot, corresponding to 0.10 and 1.00 ng/microL of clozapine in human serum after extraction process and applying 10 microL to the chromatographic plates. The method correlation coefficient was 0.999. The intra-assay variation was between 2.10 and 3.33% (n = 5) and the interassay was between 2.67 and 4.44% (n = 9). The detection limit was 0.03 ng/microL, and the quantification limit was 0.05 ng/microL. The method proved to be accurate, with a recovery between 97.00 and 99.00%, with an RSD not higher than 7.22%, and was selective for the active principle tested. This method was successfully applied to quantify clozapine in patient serum samples. In conclusion, the method is useful for the quantitative determination of clozapine in serum.     

小白菜中残留虫酰肼的超临界流体萃取条件的研究

将超临界流体萃取(supercritical fluid extraction,SFE)技术与高效液相色谱分析相结合,建立了特异性杀虫剂虫酰肼的萃取分离方法。SFE对虫酰肼的萃取条件:压力48.3 MPa(7000 psi),温度60 ℃,静态萃取时间20 min,CO2体积10 mL,改性剂甲醇添加量0.04 mL/g,丙酮为收集溶剂。在此条件下,SFE对虫酰肼的萃取率为100.75%,所得样品可直接用于高效液相色谱分析。色谱条件：紫外-二极管阵列检测器(检测波长为245 nm),C18键合色谱柱,乙腈     

Multielement electrothermal atomic absorption spectrometry: A study on direct and simultaneous determination of chromium and manganese in urine

A study was carried out on the direct determination of Cr and Mn in urine using simultaneous atomic absorption spectrometry (SIMAAS). The heating program conditions, the absorbance signal profiles, the influence of different chemical modifiers, and the urine sample volume delivery into the tube were optimized to perform the calibration with aqueous solutions. Among several chemical modifiers tested, the best recovery and repeatability results were obtained for 3 microg Mg(NO3)2. On using this modifier, the pyrolysis and atomization temperatures for simultaneous determination of Cr and Mn were 1,300 degrees C and 2,500 degrees C, respectively. Urine samples were diluted (1+1) with 2.0% (v/v) HNO3 + 0.05% (w/v) Triton X-100 prepared in high purity water. A 20-microL aliquot of analytical solution and 10 microL of chemical modifier solution were delivered to the graphite tube. The characteristic masses were 7.8 pg for Cr (RSD=4.0%) and 4.6 pg for Mn (RSD=2.6%). The limits of detection were 0.08 microg L(-1) (n=20, 3s) for Cr and 0.16 microg L(-1) (n=20, 3s) for Mn. Recovery studies for 1.0 or 2.5 microg L(-1) of Cr and Mn added to different urine samples showed acceptable results for Cr (100%, RSD=14%) and Mn (88%, RSD=5.6%).     

Optimization of supercritical fluid extraction by carbon dioxide with organic modifiers of polycyclic aromatic hydrocarbons from urban particulate matter

The main advantages of using supercritical fluids for the extractions of organic pollutants from environmental matrix is that they are inexpensive, contaminant free, and less costly to dispose safely than organic solvents. In this work, a series of extraction experiments were carried out using CO2 as supercritical fluid on a certified sample of "Urban dust" (NIST S.R.M. 1649a) to optimize the analytical parameters with the aim of investigating the extraction limit of organic pollutant by using an almost "organic solvent-free" technique. The certified sample contains small concentrations of several organic pollutants, as PAH and PCB. The initial tests of extraction were carried out with only CO2 in supercritical phase, by maintaining the temperature at 50 degrees C and 80 degrees C and by making the pressure vary between 230 bar and 600 bar. The effect of three organic modifiers (methanol, n-hexane and toluene), added at 5% in volume, has been considered. The yield of recovery has been estimated for anthracene, fluoranthene, chrysene, benzo[a]pyrene, indeno[1,2,3-cd]pyrene by GC-MS according to the increasing molecular weight.     

Application of pressurized liquid extraction followed by gas chromatography-mass spectrometry to determine 4-nonylphenols in sediments

A time- and solvent-saving method, pressurized liquid extraction (PLE), to extract 4-nonylphenol (4-NP) in sediment was developed. The effects of various operational parameters (i.e., temperature, pressure, etc.) for the quantitative extraction of 4-NP by PLE were investigated. The analytes were then identified and quantitated by a large-volume injection GC-MS technique. The 4-NP can be completely extracted by methanol at 100 degrees C and 100 atm combined with 15 min static and then 10 min dynamic extraction steps (1 atm = 101,325 Pa). Recovery of 4-NP in spiked blank kaolin samples was 98% with 5% RSD. The degrees of recovery of 4-NP in the spiked sediment samples from a reservoir and a polluted river were 111% with 4% RSD and 106% with 5% RSD, respectively. The perfect applicability of PLE for 4-NP was determined after testing it with spiked and aged samples. The extraction efficiency of the PLE was compared with conventional Soxhlet and bath ultrasonication extraction methods using the spiked sediment samples.     

Determination of polyethylene glycol in low-density polyethylene by large volume injection temperature gradient packed capillary liquid chromatography

Polyethylene glycol (PEG) 20000 in low-density polyethylene has been determined using column switching and inverse temperature programming in reversed-phase packed capillary liquid chromatography with evaporative light scattering detection. PEG 20000 was extracted into water from the polyethylene dissolved in toluene and PEG 35000 was added as an internal standard (I.S.). The samples in aliquots of 100 microl were reconcentrated on the enrichment column using a loading mobile phase of acetonitrile-water (3:97, v/v) at a flow-rate of 75 microl/min for 3 min, then back-flushed and separated on the analytical column with acetonitrile-THF-water (40:5:55, v/v) as mobile phase. The column temperature was reduced from 68 to 55 degrees C with a ramp of -1.5 degrees C/min, held constant for 3 min and then reduced further to 45 degrees C with a -1.5 degrees C/min ramp and kept constant for 1 min. The analysis runtime was 20 min. The recovery of PEG 20 000 was determined to 65.1% with 2.8% RSD and the mass limit of detection of PEG 20 000 was 1.25 microg. The within-assay and between day precision of the retention times of both PEG 20000 and PEG 35000 displayed RSD of less than 1.1% (n = 9), while the overall area ratio RSD of 100 microg/ml PEG 20000 over PEG 35000 was 1.3% (n = 9). The method was linear within the investigated concentration range 25-125 microg/ml (R2 = 0.9983). In addition, a mixture of PEG 4000, 8000, 10000, 20000 and 35000 was analysed on the system to demonstrate the possibility of analysing several PEGs in a sample with the use of temperature gradient elution.     

The use of supercritical fluid extraction for the determination of steroids in animal tissues.

A multi-analyte, multi-matrix method was developed for the routine determination of steroids in animal tissues (skin, meat and fat). After addition of internal standards and sample pre-treatment, the analytes of interest were extracted from the matrix with unmodified supercritical CO2 and trapped directly on an alumina sorbent placed in the extraction vessel (in-line trapping under supercritical conditions). After extraction, alkaline hydrolysis was performed and the analytes were derivatised. The samples were then analysed by gas chromatography-mass spectrometry. The limit of detection for the different matrix-analyte combinations was 2 micrograms kg-1 (for melengestrol acetate 5 micrograms kg-1), the repeatability ranged from 4 to 42% (n = 9) and the reproducibility ranged from 2 to 39% (n = 3).     

Determination of beryllium and selenium in human urine and of selenium in human serum by graphite-furnace atomic absorption spectrophotometry.

For human urine beryllium (Be), each sample (500 microl) was diluted (1+1) with Nash reagent (containing 0.2% (v/v) acetylacetone and 2.0 M ammonium acetate buffer at pH 6.0) and then a 20-microl volume of Triton X-100 (0.4%, v/v) aqueous solution was added. An aliquot (10 microl) of the diluted urine mixture was introduced into a graphite cuvette and was atomized according to a temperature program. The method detection limit (MDL, 3sigma) for Be was 0.37 microg/l in the undiluted urine sample and the calibration graph was linear up to 65.0 microg/l. Calibration graphs were prepared by the standard addition method. Accuracies of 98.6-102% were obtained when testing standard reference material (SRM 2670) freeze dried human urine samples. Precision (relative standard deviation, RSD) for urine Be was < or = 2.3% (withinrun, n = 5) and was < or = 3.0% (between-run, n = 3). For human urine and serum selenium (Se), samples (100 microl) were diluted with HNO3 (0.2%, v/v) to make a (1+1) dilution for urine analysis or a (1+4) dilution for serum analysis. An additional aliquot (10 microl) of Triton X-100 (0.1%, v/v) was added to each 200 microl of (1+1) diluted urine (or 20 microl of the Triton X-100 was added to each 500 microl of (1+4) diluted serum) sample. After the diluted sample mixture (10 microl) was introduced into a graphite cuvette, the corresponding chemical modifier (10 microl, containing Ni2+ + Pd + NH4NO3 in HNO3 (0.2%, v/v)) was added to it and the mixture was atomized. The MDL (3sigma) for Se in urine and in serum was 4.4 and 21.4 microg/l in undiluted sample, respectively, and the calibration graphs were linear up to 150 and 400 microg/l. Accuracies of urine Se were 98.9 - 99.4% by testing SRM 2670 (NIST) urine standards with RSD (between-run, n = 3) within 2.9%; and that of serum Se was 97.2% when testing a certified second-generation human serum (No. 29, #664) with RSD (between-run, n = 3) of 1.4%. The proposed method can be applied easily, directly, and accurately to the measurement of Be and Se in real samples (including six urine Se and four serum Se from patients of Blackfoot Disease in Taiwan).     

A two-step supercritical fluid extraction of polycyclic aromatic hydrocarbons from roadside soil samples

A two-step procedure for the supercritical fluid extraction (SFE) of polycyclic aromatic hydrocarbons from soil samples was developed. The procedure consists of a static supercritical fluid treatment in a closed extraction cell at a high temperature (T=250 or 340degreesC for 20 min) and an SFE with a solvent trapping. During the static phase, the sample is exposed to a supercritical organic solvent (methanol, toluene, dichloromethane, ACN, acetone, and hexane). The solvent penetrates particles of the matrix to substitute strongly bonded molecules and dissolves the analytes in the supercritical phase. At ambient temperature, supercritical fluids became liquid and lost their solvation abilities. Most of the analytes condense on the surface of the particles or on the extraction cell walls without forming strong bonds or penetrating deep into the matrix. Thus, the pretreatment liberates the analytes and they behave similar to those in freshly spiked samples. The common SFE with toluene-modified CO2 as an extraction fluid follows the static phase. With the use of the most suitable extraction phases (toluene, ACN), the extraction efficiency of the combined procedure is much higher (approximately100%). The results of the combined procedure are compared to the SFE procedure of the same untreated sample (difference less than 5%) and to the Soxhlet extraction. The extracts were analyzed using a GC with the flame ionization detection.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

A rapid and specific approach for direct measurement of donepezil concentration in human plasma by LC-MS/MS employing solid-phase extraction

A selective, rapid and simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for assay of donepezil in human plasma using escitalopram as an internal standard. Chromatographic separation was achieved on a Betabasic-C(8), 5 microm, 100 x 4.6 mm column using methanol:water:formic acid (90:9.97:0.03, v/v/v) as mobile phase. Detection of donepezil and internal standard was achieved by ESI MS/MS in positive ion mode using 380.20/91.10 and 325.13/262.00 transitions, respectively. The linearity over the concentration range of 0.15-50 ng/mL for donepezil was obtained and the lower limit of quantification was 0.15 ng/mL. For each level of quality control samples, inter-day and intra-day precisions (RSD) were < or =8.92 and 10.35% and accuracy (%RE) were < or =7.33% and 9.33%, respectively. The recovery was more than 88.50% for both donepezil and internal standard by solid-phase extraction, eliminating evaporation and reconstitution steps.