Highly Sensitive LC Method with Automated Co-Sense System and Fluorescence Detection for Determination of Sertraline in Human Plasma

A highly sensitive LC method with column-switching “Co-sense” system and fluorescence detection has been proposed for trace determination of sertraline in human plasma. A simple pre-column derivatization procedure with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reagent was employed. Fluxetine was used as an internal standard. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9997) was found between the peak area ratio and sertraline concentration in the range of 5–5,000 ng mL^?1. The limit of detection and limit of quantitation were 1.41 and 4.28 ng mL^?1, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 5.63%. The accuracy of the method was proved; the recovery of sertraline from the spiked human plasma was 99.76–102.62 ± 2.19–5.63%. The proposed method had high throughput as the analysis involved simple sample pre-treatment procedure and short run-time (~12 min). The results demonstrated that the method would have a great value if applied in bioavailability and pharmacokinetic studies for sertraline.     

Simultaneous Determination of Safingol and d-erythro-Sphinganine in Human Plasma by LC with Fluorescence Detection

l-threo-Sphinganine (safingol) is a putative synthetic sphingosine kinase inhibitor currently being tested in clinical trials as an anticancer agent. To enable defining the pharmacokinetic properties of safingol in humans, we developed a sensitive analytical method to simultaneously quantitate safingol and its naturally-occurring diastereomer, d-erythro-sphinganine in human plasma. Of the two different fluorogenic derivatization agents (NDA and OPA) and several pH conditions compared for the derivatization, we found that NDA derivatization achieved more than 20 times greater sensitivity compared with OPA derivatization, and pH 9.0 showed the highest sensitivity for both compounds. An analytical method for liquid chromatography (LC) with a fluorescence detector (FLD) was developed and validated with calibration curve ranges of 20–1,000 ng mL⁻¹ for safingol and d-erythro-sphinganine. Our LC-FLD method using NDA-derivatization enabled simultaneous quantification of safingol and its naturally-occurring diastereomer, d-erythro-sphinganine with satisfactory sensitivity in human plasma.

     

Simultaneous Determination of Safingol and d-erythro-Sphinganine in Human Plasma by LC with Fluorescence Detection

l-threo-Sphinganine (safingol) is a putative synthetic sphingosine kinase inhibitor currently being tested in clinical trials as an anticancer agent. To enable defining the pharmacokinetic properties of safingol in humans, we developed a sensitive analytical method to simultaneously quantitate safingol and its naturally-occurring diastereomer, d-erythro-sphinganine in human plasma. Of the two different fluorogenic derivatization agents (NDA and OPA) and several pH conditions compared for the derivatization, we found that NDA derivatization achieved more than 20 times greater sensitivity compared with OPA derivatization, and pH 9.0 showed the highest sensitivity for both compounds. An analytical method for liquid chromatography (LC) with a fluorescence detector (FLD) was developed and validated with calibration curve ranges of 20–1,000 ng mL^?1 for safingol and d-erythro-sphinganine. Our LC-FLD method using NDA-derivatization enabled simultaneous quantification of safingol and its naturally-occurring diastereomer, d-erythro-sphinganine with satisfactory sensitivity in human plasma.     

Determination of Penciclovir in Human Plasma Using LC with Fluorescence Detection: Application to a Bioequivalence Study

A novel HPLC method with fluorescence detection and one step sample preparation was developed for the determination of penciclovir in human plasma. Plasma samples (200 μL) were deproteinized by precipitation with 100 μL of perchloric acid and centrifuged. Separation was on an Inertsil ODS-3 column with a mixture of methanol–0.1% orthophosphoric acid as mobile phase. The fluorescence detector was set at Ex 270 nm, Em 375 nm. The assay was selective and linear with a limit of quantification of 0.025 mg L^?1. The mean absolute recovery was 98.1%, while the intra- and inter-day coefficients of variation and percent error values of the assay method were all less than 10%. The assay was successfully applied in a randomized cross-over bioequivalence study of three pharmaceutical products containing 250 mg famciclovir (an oral prodrug of penciclovir) in 18 healthy volunteers.     

Analysis of Amisulpride in Human Plasma by SPE and LC with Fluorescence Detection

A rapid, precise, accurate, and selective high-performance liquid chromatographic method with fluorescence detection has been validated and used for analysis of amisulpride in human plasma after a simple solid-phase extraction procedure. Compounds were separated on a CN column with 0.03?M potassium dihydrogen phosphate (pH 6.5)-acetonitrile 65:35 (v/v) as mobile phase. Fluorescence detection was performed at excitation and emission wavelengths of 274 and 370?nm, respectively. Calibration plots were linear over the concentration range 10-1,000?ng?mL(-1) in human plasma, and the lower limit of quantification was 10?ng?mL(-1). Accuracy was between 0.4 and 6.4% and precision was between 3.1 and 7.5%. Amisulpride was sufficiently stable through three freeze-thaw cycles, during storage for 6?h at room temperature, and for 2?months at -22?°C. The method is suitable for the analysis of clinical samples from pharmacokinetic studies.     

Sensitive, Selective, Precise and Accurate LC–MS Method for Determination of Clonidine in Human Plasma

A selective and sensitive liquid chromatography–mass spectrometric method in ESI (+ve) mode was developed and validated completely in human plasma. Clonidine and IS, carbamazapine, were extracted from human plasma via liquid–liquid extraction with ethyl acetate. Following evaporation under nitrogen, the residue was reconstituted with mobile phase and analyzed using API 4000 LC–MS–MS system. An isocratic program with binary mobile phases (0.1% formic acid in water and acetonitrile) was used to separate interference peaks by a C18 analytical column. Linearity range was 0.49–73.98 ng mL^?1. The intra- and inter-day accuracy and precision were within acceptable limits (≤15%). This method was successfully applied to a single dose 25 μg tablets BE study of clonidine in healthy male volunteers.     

Sensitive,Selective, Precise and Accurate LC–MS Method for Determination of Clonidine in Human Plasma

A selective and sensitive liquid chromatography–mass spectrometric method in ESI (+ve) mode was developed and validated completely in human plasma. Clonidine and IS, carbamazapine, were extracted from human plasma via liquid–liquid extraction with ethyl acetate. Following evaporation under nitrogen, the residue was reconstituted with mobile phase and analyzed using API 4000 LC–MS–MS system. An isocratic program with binary mobile phases (0.1% formic acid in water and acetonitrile) was used to separate interference peaks by a C18 analytical column. Linearity range was 0.49–73.98 ng mL⁻¹. The intra- and inter-day accuracy and precision were within acceptable limits (≤15%). This method was successfully applied to a single dose 25 μg tablets BE study of clonidine in healthy male volunteers.

     

Rapid RP-LC Method with Fluorescence Detection for Analysis of Fexofenadine in Human Plasma

A rapid and sensitive reversed-phase high-performance liquid chromatographic method for analysis of fexofenadine in human plasma has been developed and optimized. The analytes were extracted from biological samples by solid-phase extraction on hydrophilic–lipophilic balance cartridges. LC separation was performed on a C₁₈ analytical column (125 mm × 4 mm i.d., 5-μm particles) with 42:58 (v/v) acetonitrile–water adjusted to pH 2.7 with 85% orthophosphoric acid as mobile phase. Fluorescence detection was performed with excitation at 230 nm and emission at 290 nm. The total time for chromatographic separation was 7 min. The method was validated in accordance with EU guidelines by analysis of plasma samples fortified with fexofenadine at concentrations between 0.05 and 800 ng mL⁻¹. Calibration plots were linear in this range. Mean recovery was typically 94.03% and the detection limit was 0.05 ng mL⁻¹. The time required for quantitative analysis is shorter than that required by other methods.

     

Rapid RP-LC Method with Fluorescence Detection for Analysis of Fexofenadine in Human Plasma

A rapid and sensitive reversed-phase high-performance liquid chromatographic method for analysis of fexofenadine in human plasma has been developed and optimized. The analytes were extracted from biological samples by solid-phase extraction on hydrophilic–lipophilic balance cartridges. LC separation was performed on a C₁₈ analytical column (125 mm × 4 mm i.d., 5-μm particles) with 42:58 (v/v) acetonitrile–water adjusted to pH 2.7 with 85% orthophosphoric acid as mobile phase. Fluorescence detection was performed with excitation at 230 nm and emission at 290 nm. The total time for chromatographic separation was 7 min. The method was validated in accordance with EU guidelines by analysis of plasma samples fortified with fexofenadine at concentrations between 0.05 and 800 ng mL^?1. Calibration plots were linear in this range. Mean recovery was typically 94.03% and the detection limit was 0.05 ng mL^?1. The time required for quantitative analysis is shorter than that required by other methods.     

Quantitative Determination of Phillyrin in Human Plasma Using HPLC with Fluorescence Detection

A simple, rapid, and selective high-performance liquid chromatography method for determination of phillyrin in human plasma was developed. After extracting from the plasma samples with ethyl acetate, the analyte was chromatographed on a C18 column with methanol–water (50:50, v/v, pH 2.86) as mobile phase. The fluorescence excitation and emission wavelengths were 277 and 315 nm, respectively. The linear range of the standard curve of phillyrin was 0.0313–8.0 μg mL^?1 (r > 0.999). The limit of detection was 6.31 ng mL^?1. The average recovery of phillyrin was 101.02% from plasma. The intra- and inter-day variabilities of phillyrin were <10.00%.     

LC Determination of Amino Acids in Rat Plasma with Fluorescence Detection: Application to Exercise Physiology

An LC method for the determination of 20 amino acids (AAs), using 1,2-Benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) as fluorescent labeling reagent, has been validated and applied for the analysis of AAs in rat plasma at three different states concerning exercise physiology. Identification of AA derivatives was carried out by LC-MS with electrospray ion (ESI), and the MS-MS cleavage mode of the representative tyrosine (Tyr) derivative was analyzed. Gradient elution on a Hypersil BDS C₁₈ column gave good separation of the derivatives. Excellent linear responses were observed and good compositional data could be obtained from as little as 50–200 μL of plasma samples. The contents of 20 AAs in rat plasma of three groups (24 rats, group A: quiet state, group B: at exercising exhaust, group C: 12 h after exercising exhaust) exhibited evident difference corresponding to the physiological states. Facile BCEOC derivatization coupled with LC-FLD-ESI-MS analysis allowed the development of a highly sensitive method for the quantitative analysis of trace level of AAs from plasma or other biochemical samples.     

毛细管电泳-激光诱导荧光法测定人血浆中的富马酸比索洛尔

建立了毛细管电泳-激光诱导荧光检测(CE-LIFD)技术测定人血浆中富马酸比索洛尔含量的新方法。选用荧光素异硫氰酸酯(FITC)为衍生化试剂,当缓冲溶液为25mmol/LNa2B4O7(pH9.2)、分离电压25kV、柱温20℃、电动进样(10kV×8s)、以峰面积内标法定量、于激发波长/发射波长=488/520nm柱上检测时,富马酸比索洛尔得到较好分离。在选定的电泳条件下,对其线性范围(20～1000μg/L)、检出限(10μg/L)、重现性(日内和日间精密度分别小于4.44%和5.59%)和回收率(96.19%～101.80%)进行了测定。结果表明:迁移时间的重现性<1.58%;峰面积之比的重现性<6%。     

A Rapid Determination of Taurine in Human Plasma by LC

Taurine is an amino acid which is not incorporated into proteins but found in the cytosol of many mammalian cells, in high concentrations (2–30 mM). Increase in plasma taurine concentration has already been reported after surgical trauma, X-radiation, muscle necrosis, carbon tetrachloride-induced liver damage, and paracetamol overdose. Plasma taurine concentration was measured using LC with fluorescence detection following derivatization by o-phtalaldehyde plus 3-mercapto-propionic acid and α-aminobutyric acid as internal standard. Under these conditions the retention time of taurine was 10 min. This method was sensitive enough, to quantify 150 pg mL^?1 and detect 50 pg mL^?1 of taurine ranging normally between 65 and 179 mmol L^?1 (8–22 μg mL^?1). The validated method allowed simple determination of human plasma taurine in pharmacokinetic and biomarker studies.     

A Rapid Determination of Taurine in Human Plasma by LC

Taurine is an amino acid which is not incorporated into proteins but found in the cytosol of many mammalian cells, in high concentrations (2–30 mM). Increase in plasma taurine concentration has already been reported after surgical trauma, X-radiation, muscle necrosis, carbon tetrachloride-induced liver damage, and paracetamol overdose. Plasma taurine concentration was measured using LC with fluorescence detection following derivatization by o-phtalaldehyde plus 3-mercapto-propionic acid and α-aminobutyric acid as internal standard. Under these conditions the retention time of taurine was 10 min. This method was sensitive enough, to quantify 150 pg mL⁻¹ and detect 50 pg mL⁻¹ of taurine ranging normally between 65 and 179 mmol L⁻¹ (8–22 μg mL⁻¹). The validated method allowed simple determination of human plasma taurine in pharmacokinetic and biomarker studies.

     

LC with Coulometric Detection for Analysis of 5-Methyltetrahydrofolate in Human Plasma

An isocratic high-performance liquid chromatographic method with coulometric electrochemical detection has been used for analysis of 5-methyltetrahydrofolate (5-MTHF) in human plasma. A 250 mm × 4.6 mm i.d., 5-μm particle, C₁₈ column was used with 12:88 (v/v) acetonitrile ?35 mM sodium phosphate buffer pH 3.8 as mobile phase at a flow rate of 1.0 mL min^?1. The method was validated for 5-MTHF plasma concentrations in the range 2.5–100.0 nM. The method was characterized by a good linearity (regression coefficient r ≥ 0.9989) and limits of detection and quantification of 0.72 and 2.16 nM, respectively. Mean recovery at low and high concentrations ranged from 89.1 to 96.3%, respectively, with a relative standard deviation <4.6%. Between-run imprecision (4.2%) was higher than within-run imprecision (3.4%). The proposed separation and detection procedures were successfully applied to analysis of 5-MTHF in human plasma.     

Determination of the Active Metabolite of Prulifloxacin in Human Plasma by HPLC with Fluorescence Detection

A cheap, simple and rapid sample preparation method has been developed for quantification of ulifloxacin, the active metabolite of prulifloxacin in human plasma, by HPLC with fluorescence detection using lemefloxacin as the internal standard. One-step protein precipitation with 10% perchloric acid (2:1, v/v) on a 200 μL sample was used. The separation was performed at 30 °C on a C18 column using an eluent of acetonitrile-0.5% triethylamine buffer. The compounds were monitored at λ _ex of 280 nm, λ _em of 425 nm. The calibration curve for ulifloxacin in human plasma was linear over the range 0.01–1.00 μg mL⁻¹. The lower limit of quantification is 0.01 μg mL⁻¹. The intra- and inter-day precision ranged from 3.0 to 6.7%, respectively. The method had been used for clinical pharmacokinetic studies of prulifloxacin formulation product after oral administration to healthy volunteers. Jun Wen and Zhenyu Zhu have equal contribution to this work.     

Determination of Three Nonsteroidal Anti-Inflammatory Drugs in Human Plasma by LC Coupled with Chemiluminescence Detection

A simple and sensitive method was developed for the determination of three nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, naproxen and fenbufen in human plasma. The method involved in column liquid chromatographic separation and chemilumenescence (CL) detection based on the CL reaction of NSAIDs, potassium permanganate (KMnO₄) and sodium sulfite (Na₂SO₃) in sulfuric acid (H₂SO₄) medium. The chromatographic separation was carried out using a reversed-phase C₁₈ column, which allowed the selective determination of the three medicines in the complicated samples. The special features of the CL detector provided lower LOD for determination than that of existing chromatographic alternatives. The results indicated that the linear ranges were 0.01–10.0 μg mL⁻¹ for ibuprofen, 0.001–1.0 μg mL⁻¹ for naproxen, and 0.01–10.0 μg mL⁻¹ for fenbufen. The limits of detection were 0.5 ng mL⁻¹ for ibuprofen, 0.05 ng mL⁻¹ for naproxen and 0.5 ng mL⁻¹ for fenbufen (S/N = 3). All average recoveries were in the range of 90.0–102.3%. Finally, the method had been satisfactorily applied for the determination of ibuprofen, naproxen and fenbufen in human plasma samples.

     

Determination of Three Nonsteroidal Anti-Inflammatory Drugs in Human Plasma by LC Coupled with Chemiluminescence Detection

A simple and sensitive method was developed for the determination of three nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, naproxen and fenbufen in human plasma. The method involved in column liquid chromatographic separation and chemilumenescence (CL) detection based on the CL reaction of NSAIDs, potassium permanganate (KMnO₄) and sodium sulfite (Na₂SO₃) in sulfuric acid (H₂SO₄) medium. The chromatographic separation was carried out using a reversed-phase C₁₈ column, which allowed the selective determination of the three medicines in the complicated samples. The special features of the CL detector provided lower LOD for determination than that of existing chromatographic alternatives. The results indicated that the linear ranges were 0.01–10.0 μg mL^?1 for ibuprofen, 0.001–1.0 μg mL^?1 for naproxen, and 0.01–10.0 μg mL^?1 for fenbufen. The limits of detection were 0.5 ng mL^?1 for ibuprofen, 0.05 ng mL^?1 for naproxen and 0.5 ng mL^?1 for fenbufen (S/N = 3). All average recoveries were in the range of 90.0–102.3%. Finally, the method had been satisfactorily applied for the determination of ibuprofen, naproxen and fenbufen in human plasma samples.     

Sensitive LC–ESI–MS Method for Determination of Tiopronin in Human Plasma

A sensitive liquid chromatographic–electrospray ionization–mass spectrometric (LC–MS) method has been developed for direct measurement of the concentration of tiopronin in human plasma. Hydrochloric acid solution was used to stabilize the tiopronin and prevent formation of a dimer, or reaction with endogenous thiols. The method involved liquid–liquid extraction of tiopronin from plasma samples with ethyl acetate, simple reversed-phase chromatography, and mass spectrometric detection with nanogram detection limits. Acetaminophen was used as internal standard (IS). The limit of quantification was 5 ng mL^?1 (RSD 4.3%). The method was validated within the linear range 5–500 ng mL^?1. The correlation coefficient for the calibration regression line was 0.9997 or better. Intra-day and inter-day accuracy were better than 15%. The method has been successfully used for a pharmacokinetic study with human subjects. Among the pharmacokinetic data obtained, t _1/2 was 2.37 ± 0.63 h and T _max was 4 h.     

An Improved Method for the Determination of Reserpine in Plasma Using Liquid Chromatography with Fluorescence Detection

Abstract

A liquid chromatographic method coupled with fluorescence detection was developed to measure plasma reserpine concentrations. After extraction from 3 ml of plasma, the reserpine and internal standard (methyl-18-triethoxy benzoyl reserpate) residues were oxidized to their respective fluorophors by vanadium pentoxide and chromatographed on a reversed phase trimethylsilyl column. These compounds were detected at excitation wave length 460 nm and analyzed at 570 nm. The minimum quantifiable level was ca 0.3 ng/ml and the absolute recovery was determined to be between 78–83%. The coefficient of variation was less than 9% for day-to-day and within run analyses. This method is suitable for pharmacokinetic studies of reserpine in man.