Determination of valproic acid (2-propylpentanoic acid) in human plasma by capillary electrophoresis with indirect UV detection

A rapid capillary zone electrophoresis method with indirect UV detection was developed and validated for the determination of valproic acid (VPA) in human plasma. The analyses were carried out under optimized conditions, using a buffer system composed of 15 mM benzoate and 0.5 mM cetyltrimethylammonium bromide at pH 6.0, and 25% v/v methanol; 2-hydroxybutyric acid was selected as the internal standard (IS). The capillary electrophoresis (CE) separation was carried out at a negative potential of 30 kV and the indirect UV detection was operated at 210 +/- 20 nm for all assays. The influence of buffer pH, ionic strength, concentration of electroosmotic flow (EOF) modifier and organic modifier on indirect signal response and migration behavior of the organic acid was investigated. Isolation of VPA from plasma was accomplished by a carefully implemented procedure using methanol as the precipitant agent. Using a high ratio of methanol to plasma for deproteinization (4:1), good absolute recovery of the analyte and satisfactory selectivity was obtained. The calibration line for VPA was linear over the 1-100 microg/mL concentration range. Sensitivity was high; in fact, the limit of detection (LOD) of VPA was 150 ng/mL and 450 ng/mL the limit of quantitation (LOQ). The results obtained analyzing real plasma samples from schizophrenic patients under polytherapy with VPA as well as antipsychotic drugs were satisfactory in terms of precision, accuracy and sensitivity.     

Simultaneous determination of aripiprazole and its active metabolite,dehydroaripiprazole, in plasma by capillary electrophoresis combining on‐column field‐amplified sample injection and application in schizophrenia

A sensitive high‐performance CZE combining on‐column field‐amplified sample injection (FASI) has been developed for simultaneous determination of aripiprazole and its active metabolite, dehydroaripiprazole, in human plasma. A sample pretreatment by means of liquid–liquid extraction (LLE) (diethyl ether) with subsequent quantitation by FASI‐CZE was used. The separation of aripiprazole and dehydroaripiprazole was performed using a BGE containing 150 mM phosphate buffer (pH 3.5) with 40% methanol and 0.02% PVA as a dynamic coating to reduce interaction of analytes with the capillary wall. Before sample loading, a methanol plug (0.3 psi, 6 s) was injected to permit FASI for stacking. The samples were injected electrokinetically (10 kV, 30 s) to introduce sample cations and the applied voltage was 20 kV with on‐column detection at 214 nm. Several parameters affecting the separation and sensitivity of the drug and its active metabolite were studied, including reconstitution solvent, organic modifier, pH and concentration of phosphate buffer. The linear ranges of the method for test drug and its active metabolite, in plasma using amlodipine as an internal standard, were over the range 5.0–100.0 ng/mL. One female volunteer (25 years old) was orally administered a single dose of 10 mg aripiprazole (Abilify^®, Otsuka) and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied to monitor the concentration of aripiprazole and dehydroaripiprazole in plasma collected after oral administration of 20 or 30 mg aripiprazole (Abilify^®, Otsuka) daily at steady state in one schizophrenic patient.     

Validated HPLC method for the determination of fluconazole in human plasma

A high-performance liquid chromatographic assay with UV detection was developed for the determination of fluconazole in human plasma. The method utilized solid-phase extraction for sample clean-up. The separation was performed on a C18 column by isocratic elution with a mobile phase of 10 mM acetate buffer at pH 5.0 and methanol and UV detection at 210 nm. Validation was performed according to the current recommendations of the USFDA bioanalytical method validation guidance. The method proved to be specific, accurate, precise and linear between 200 and 10,000 ng/mL with correlation coefficients greater than 0.999. The coefficient of variation was within 11% and relative deviation was less than 10%.     

A validated HPLC method with electrochemical detection for simultaneous assay of 5-aminosalicylic acid and its metabolite in human plasma

A high-performance liquid chromatographic method was developed, validated and applied to the simultaneous determination of 5-aminosalicylic acid (5-ASA) and its acetylated metabolite (acetyl-5-ASA) in human plasma. The method involves liquid-liquid extraction with methanol followed by isocratic reversed-phase chromatography on a Kromasil KR100 C(18) column with electrochemical detection. The recovery, selectivity, linearity, precision and accuracy of the method were evaluated from spiked human plasma samples. The effects of mobile phase composition, buffer concentration, mobile phase pH and concentration of organic modifiers on retention of 5-ASA, acetyl 5-ASA and internal standard were investigated. Limits' of detection were 5 ng/mL for 5-ASA and 10 ng/mL for acetyl-5-ASA, respectively. The method can be used for supporting therapeutical drug monitoring and pharmacokinetic studies.     

Direct and sensitive analysis of methamphetamine, ketamine, morphine and codeine in human urine by cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography

Cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-Sweep-MEKC) was directly used to test some abuse drugs in human urine, including morphine (M), codeine (C), ketamine (K) and methamphetamine (MA). First, phosphate buffer (50 mM, pH 2.5) containing 30% methanol was filled into uncoated fused silica capillary (40 cm, 50 microm I.D.), then high conductivity buffer (100 mM phosphate, 6.9 kPa for 99.9 s) was followed. Electrokinetic injection (10 kV, 500 s) was used to load samples and to enhance sensitivity. The stacking step and separation were performed at -20 kV and 200 nm using phosphate buffer (25 mM, pH 2.5) containing 20% methanol and 100 mM sodium dodecyl sulfate. Using CSEI-Sweep-MEKC, the analytes could be simultaneously analyzed and have a detection limit down to ppb level. It was unnecessary to have sample pretreatments. During method validation, calibration plots were linear (r>or=0.9982) over a range of 150-3,000 ng/mL for M and C, 250-5,000 n g/mL for MA, and 50-1,000 ng/mL for K. The limits of detection were 15 ng/mL for M and C, and 5 ng/mL for MA and K (S/N=3, sampling 500 s at 10 kV). Comparing with capillary zone electrophoresis, the results indicated that this stacking method could increase 6,000-fold sensitivity for analysis of MA. Our method was applied for analysis of 28 real urine samples. The results showed good coincidence with immunoassay and GC-MS. This method was feasible for application to detect trace levels of abused drugs in forensic analysis.     

Simultaneous determination of cefepime and vancomycin in plasma and cerebrospinal fluid by MEKC with direct sample injection and application for bacterial meningitis

A simple MEKC with UV detection at 214 nm for simultaneous analysis of cefepime and vancomycin in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of cefepime and vancomycin from biological matrices was performed at 25 degrees C using a BGE consisting of a Tris buffer with SDS and methanol as the electrolyte solution. Under optimal MEKC conditions for biological samples, good separations with high efficiency and short analysis time are achieved. Several parameters affecting the separation of the drugs from biological matrices were studied, including methanol, pH, and concentrations of the Tris buffer and SDS. The linear ranges of the method for the determination of cefepime and vancomycin in plasma and in CSF using imidazole or cefazolin as an internal standard, respectively, were all over the range of 1-30 microg/mL; the detection limits of cefepime and vancomycin in biological matrices (injection 10 kV, 15 s) were 0.3 and 0.5 microg/mL, respectively. The applicability of the proposed method for the determination of cefepime and vancomycin in plasma and CSF collected after intravenous administration of the drugs in patients with meningitis was demonstrated.     

HPLC-DAD determination of plasma levels of the antipsychotic risperidone and its main metabolite for toxicological purposes

A new, rapid analytical method, based on liquid chromatography with diode array detection, has been developed and applied to the determination of risperidone and its main active metabolite 9-hydroxyrisperidone in human plasma. The chromatographic separation was obtained on a C8 (150 x 4.6 mm, 5 microm) column, using a mobile phase composed of acetonitrile (27%) and a pH 3.0 phosphate buffer (73%). A sample clean-up procedure was carried out by using C8 cartridges and eluting the analytes with methanol. The extraction yield was highly satisfactory for both analytes, with average absolute recovery values of about 95%. The experimental conditions permitted the quantitative determination of risperidone and 9-hydroxyrisperidone with high precision (RSD < 3.6%) and satisfactory sensitivity (LOQ = 4 ng mL(-1)). The method was applied to plasma samples from a patient who had tried to poison himself with 150 mg of risperidone, and was undergoing polypharmacy.     

Quantitation of oxcarbazepine and its metabolites in human plasma by micellar electrokinetic chromatography

A reliable micellar electrokinetic chromatographic method for the determination of oxcarbazepine and its two main metabolites, 10-hydroxycarbamazepine and 10,11-trans-dihydroxy-10,11-dihydroxycarbamazepine, in human plasma was developed. The separation and determination of the analytes was achieved using a system consisting of 60 mM SDS in phosphate buffer (30 mM, pH 8.0), to which 20% (v/v) methanol was added. Separation was carried out in an uncoated fused-silica capillary with a separation voltage of 25 kV and currents typically less than 40 microA. Spectrophotometric detection was at 205 nm. Isolation of oxcarbazepine and its metabolites from plasma was accomplished by a solid-phase extraction procedure. The mean extraction yield of the analytes from plasma was higher than 94%. The linear correlation coefficients were better than 0.994 for all analytes. The limit of detection was 0.05 microg/mL, the limit of quantitation 0.15 microg/mL. The repeatability for the spiked blank plasma samples was lower than 1.9% and the intermediate precision lower than 2.1%, both expressed as RSD%. The results obtained analysing real plasma samples from epileptic patients under therapy with Tolep were satisfactory in terms of precision, accuracy and detectability.     

Analysis of alendronate in human urine and plasma by magnetic solid-phase extraction and capillary electrophoresis with fluorescence detection

A simple, rapid and sensitive CE-fluorescence (FL) detection method for the analysis of alendronate (ALEN), a bisphosphonate drug, has been developed. Using a buffer solution of 20 mM sodium phosphate (pH 10.0) and a voltage of 24 kV, separation of ALEN in a 55-cm length (35-cm effective length) capillary was achieved in 5 min. FL detection of ALEN was performed via pre-column derivatization with 2,3-naphthalene dicarbox-yaldehyde (NDA). Linear correlation (r=0.9981, n=6) between FL intensity and analyte concentration was obtained in the range of 7-200 ng/mL ALEN. The developed CE-FL method was applied to the analysis of ALEN in human urine and plasma samples. In order to eliminate the interfering matrix components, SPE using magnetic Fe(3) O(4) @Al(2) O(3) nanoparticles as solid sorbents was employed to clean the biological fluids before CE-FL analysis. The linear ranges of ALEN in urine and plasma were 5-100 ng/mL (r = 0.9982, n = 7) and 5-70 ng/mL (r = 0.9954, n = 7), respectively. The LOD and LOQ in both urine and plasma samples were 1.5 and 5 ng/mL ALEN, respectively. Total analysis time including sample pre-treatment and CE separation was less than 1.5 h.     

Determination of cefepime in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection

A simple micellar capillary electrokinetic chromatography (MEKC) with UV detection is described for analysis of cefepime in plasma and cerebrospinal fluid by direct injection without any sample pretreatment. The separation of cefepime from biological matrix was performed at 25 degrees C using a background electrolyte consisting of tris(hydroxymethyl)aminomethane (Tris) buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Several parameters affecting the separation of the drug were studied, including the pH and concentrations of the Tris buffer and SDS. Using cefazolin as an internal standard, the linear ranges of the method for the determination of cefepime in plasma and cerebrospinal fluid were 1-50 and 1-20 microg/mL, respectively; the detection limits of plasma (signal-to-noise ratio = 3; injection, 5 kV, 5 s) and cerebrospinal fluid (signal-to-noise ratio = 3; injection, 0.5 psi, 3 s) were 0.2 microg/mL and 0.3 microg/mL, respectively. Application of the proposed method for determination of cefepime in plasma and cerebrospinal fluid collected after intravenous administration of 2 g cefepime in patients with meningitis was demonstrated.     

反相高效液相色谱法同时测定人血浆中的西诺沙星和萘啶酸

提出了同时测定人血浆中的西诺沙星和萘啶酸的高效液相色谱方法。采用反相C18柱、254nm紫外检测,乙腈－甲醇－0．01mol/L草酸（30：5：65,V／V;pH4．55）体系,实现了二者的良好分离。分别考察了磷酸缓冲溶液和草酸缓冲溶液作为流动相弱溶剂,结果草酸缓冲溶液能有效改善峰形、降低检测限。在不同pH值（pH2．80,4．08,4．55）进行了实验,pH4．55获得最佳分离,最低检测量分别为0．14ng（西诺沙星）和0．24ng（萘啶酸）,比文献报道的同类药物最低检测量低10倍。     

Sensitive analysis of donepezil in plasma by capillary electrophoresis combining on-column field-amplified sample stacking and its application in Alzheimer's disease

Field-amplified sample stacking (FASS) in capillary electrophoresis (CE) was used to determine the concentration of donepezil, an acetylcholinesterase inhibitor, in human plasma. A sample pretreatment by liquid-liquid extraction with isopropanol/n-hexane (v/v 3:97) and subsequent quantification by FASS-CE was used. Before sample loading, a water plug (0.5 psi, 6 s) was injected to permit FASS. Electrokinetic injection (7 kV, 90 s) was used to introduce sample cations. The separation condition for donepezil was performed in electrolyte solutions containing Tris buffer (60 mM, pH 4.0) with sodium octanesulfonate 40 mM and 0.01% polyvinyl alcohol as a dynamic coating to reduce analytes' interaction with capillary wall. The separation was performed at 28 kV and detected at 200 nm. Using atenolol as an internal standard, the linear ranges of the method for the determination of donepezil in human plasma were over a range of 1-50 ng/mL. The limit of detection was 0.1 ng/mL (S/N=3, sampling 90 s at 7 kV). One female volunteer (54 years old) was orally administered a single dose of 10 mg donepezil (Aricept, Eisai), and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied successfully to monitor donepezil in sixteen Alzheimer's disease patients' plasmas.     

Validation study of assay method for DX-8951 and its metabolite in human plasma and urine by high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

A new liquid chromatographic/mass spectrometric assay has been developed for the determination of DX-8951, a new anti-tumor drug, and its 4-hydroxymethyl metabolite (UM-1) in human plasma and urine. Solid-phase extractions were used for sample preparation. A gradient reverse-phase HPLC separation was developed with mobile phases consisting of trifluoroacetic acid and methanol. The detection was conducted using atmospheric pressure chemical ionization tandem mass spectrometry in the selected reaction monitoring mode. A structural analog, camptothecin (CPT), was used as the internal standard. The assay was validated for the determination of DX-8951 and UM-1 in human plasma and urine. The lower limits of quantitation of DX-8951 and UM-1 were 0.1 ng/mL in plasma and 1 ng/mL in urine. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.     

毛细管电泳电致化学发光检测血浆中布比卡因

布比卡因是一种外科局部麻醉剂,使用过量会导致中枢神经系统和心脏血管系统中毒^[1],可引起心脏停博.高效液相色谱和毛细管电泳(CE)^[2]是该药常用的检测方法.     

HPLC method for determination of diclofenac in human plasma and its application to a pharmacokinetic study in Turkey

A simple high-performance liquid chromatography (HPLC) method has been developed for determination of diclofenac in human plasma. The method was validated on Ace C(18) column using UV detection. The mobile phase consisted of 20 mM phosphate buffer (pH 7) containing 0.1% trifluoroacetic acid-acetonitrile (65:35, v/v). Calibration curve was linear between the concentration range of 75-4000 ng/mL. Intra- and inter-day precision values for diclofenac in plasma were less than 3.6, and accuracy (relative error) was better than 5.3%. The limits of detection and quantification of diclofenac were 25 and 75 ng/mL, respectively. Also, this assay was applied to determine the pharmacokinetic parameters of diclofenac in healthy Turkish volunteers who had been given 50 mg diclofenac.     

Micellar electrokinetic capillary chromatography as a powerful tool for pharmacological investigations without sample pretreatment: a precise technique providing cost advantages and limits of detection to the low nanomolar range.

A number of pharmaceuticals (e.g., acetaminophen, salicylic acid, sulfamethoxazole, theophylline, tolbutamide and trimethoprim) have been determined in human plasma by micellar electrokinetic chromatography (MEKC), without sample pretreatment, using underivatized fused-silica capillaries. The total analysis time was only 10 min. A sodium dodecyl sulfate (SDS)-containing borate buffer (60 mM with 200 mM SDS) at pH 10 was used. Between runs, proteins adsorbed to the capillary wall are removed by rinsing with SDS buffer and either acetonitrile (e.g., 50% v/v) or isopropanol (e.g., 10% v/v). Other rinsing procedures are discussed (salts, enzyme-containing solutions, organic solvents, sodium hydroxide, hydrofluoric acid). The separation system is tested in a concentration range between 10 ng/mL and 100 microg/mL; a detection limit of about 20 ng/mL can readily be obtained. The sensitivity was substantially improved using isopropanol as buffer additive. A day-to-day precision for relative peak areas of 1-2% relative standard deviation (RSD, n > 40) was reached in the upper concentration range. Under repeatability conditions, these values could also be obtained for low microg/mL concentrations. Thus, not only drug monitoring but also pharmacokinetic investigations from blood plasma become possible without further sample pretreatment.     

Separation and determination of closely related lantibiotics by micellar electrokinetic chromatography

A sensitive micellar electrokinetic chromatography (MEKC) method was developed for the separation and determination of four closely related lantibiotics: gallidermin, cinnamycin, duramycin and nisin. Factors affecting the separation of the lantibiotics such as pH, phosphate buffer concentration, SDS concentration and wavelength for UV detection were investigated. By optimizing these experimental conditions, successful separation was achieved between class 1A lantibiotics (nisin and gallidermin) and class 1B lantibiotics (duramycin and cinnamycin). The four lantibiotics were separated within 12 min in 50 mM phosphate buffer at pH 3.95 ± 0.1 containing 80 mM SDS with UV detection of 214 nm. The LOD (S/N = 3) were 61 ng/mL for gallidermin, 57 ng/mL for cinnamycin, 55 ng/mL for duramycin and 58 ng/mL for nisin. The method was successfully applied to real samples such as fermentation broth, bovine colostrum and predrop beer. This method yielded satisfactory results, with quantitative recoveries of spiked lantibiotics in the three samples ranging from 86.1 to 99.6%.     

胶束扫集毛细管电泳分离测定绿原酸和咖啡酸

采用胶束扫集毛细管电泳分离测定双黄连口服液中的绿原酸和咖啡酸.试验条件为:重力进样时间40 s;以20 mmol/L NaH_2PO_4,100 mmol/L 十二烷基磺酸钠(SDS)为电泳缓冲液(含体积分数15%甲醇,pH 2.20),分离电压-20 kV,检测波长214 nm,讨论了pH、SDS浓度、样品溶剂等对分离效果的影响.在优化条件下,绿原酸和咖啡酸的检出限分别达到1.02和0.168 μg/mL,线性范围分别为5.86～51.5 μg/mL和1.27～14.5 μg/mL.     

Determination of morphine, codeine, and paclitaxel in human serum and plasma by micellar electrokinetic chromatography

A micellar electrokinetic chromatography method is proposed for the determination of morphine, codeine, and paclitaxel at clinical relevant levels in human serum and plasma, which are employed in the treatment of patients with cancer. Optimal conditions for the separation were investigated. A background electrolyte solutions consisting of 20 mM borate buffer adjusted to pH 8.5, sodium dodecyl sulphate 60 mM and 15% methanol, hydrodynamic injection, and 25 kV as separation voltage were used. Detection wavelength was 212 nm for morphine and codeine and 200 nm for paclitaxel. Aspects such as stability of the solutions, linearity, accuracy, precision, and robust and ruggedness were examined in order to validate the proposed method. Detection limits obtained for all the studied compounds ranged between 26 and 52 ng/mL. Before micellar electrokinetic chromatography determination, the samples were purified and enriched by means of an extraction-preconcentration step with a preconditioned C(18) cartridge. This method was applied to the analysis of serum and plasma samples from different cancer patients undergoing treatment with paclitaxel or/and codeine.     

Rapid determination of acyclovir in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection and its clinical application

A simple MEKC with UV detection at 254 nm for analysis of acyclovir in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of acyclovir from biological matrix was performed at 25 degrees C using a BGE consisting of Tris buffer with SDS as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Using dyphylline as an internal standard, the linear ranges of the method for the determination of acyclovir in plasma and in CSF all exceeded the range of 2-50 microg/mL; the detection limit of the drug in plasma and in CSF (S/N = 3; injection 3.45 kPa, 5 s) was 1.0 microg/mL. The applicability of the proposed method for determination of acyclovir in plasma and CSF collected at 8 h after intravenous administration of 500 mg acyclovir (Zovirax) in two patients with herpes simplex encephalitis was demonstrated.