Rapid analysis of fluoxetine and its metabolite in plasma by LC-MS with column-switching approach

A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography–electrospray ionisation–mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4 min. Method performances were evaluated. The method showed good linearity in the range of 25–1000 ng mL^–1 with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25 ng mL^–1 for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma samples.     

Determination of fosfomycin in chicken plasma samples by gas chromatography: application to pharmacokinetic studies

Summary A sensitive gas chromatographic method is described for the analysis of fosfomycin (FOS) in chicken plasma using phenylphosphonic acid (PPA) as the internal standard. Plasma samples were ultrafiltered, and the ultrafiltrate was then evaporated to dryness. The residue was reconstituted with a silylation reagent for the derivatization of FOS and PPA. The methodology involves the use of a HP-5 capillary column and a flame ionisation detector (FID). The retention times of FOS and PPA were 4.63 and 8.68 minutes, respectively. Response was linear in the range of 1–150 μ mL⁻¹. The detection and quantitation limits were 1 and 2.1 μ mL⁻¹, respectively. Recovery was determined as 109% for FOS. The method was applied to the determination of FOS in chicken plasma samples collected during pharmacokinetic studies.     

Simultaneous Determination of Berberine and Palmatine in Rabbit Plasma by LC-MS–MS and Its Application in Pharmacokinetic Study After Oral Administration of Coptidis and Coptidis–Gardeniae Couple Extract

A valid and sensitive LC-MS–MS method is adopted for pharmacokinetics study of berberine and palmatine in rabbit plasma. After mixing with internal standard tetrahydroberberine, plasma samples were pretreated with 1.5 mL acetonitrile. Chromatographic separation was on a C₁₈ column using a mixture of water (containing 10 mmol L^?1 ammonium acetate, pH 3.5) and acetonitrile (50∶50, v/v) as mobile phase. The detection was performed by selected ion monitoring mode via electrospray ionization source operating in the positive ionization mode. The method was linear over the concentration range of 2.0–200.0 ng mL^?1 for berberine and 1.0–100.0 ng mL^?1 for palmatine. The lowest limits of quantitation (LLOQ) were 2.0 ng mL^?1 for berberine and 1.0 ng mL^?1 for palmatine. The intra- and inter-day precision values were less than 14.3% and the deviations were within ±11.0%. The fully validated LC-MS–MS method has been successfully applied to a pharmacokinetic study of berberine, palmatine in rabbit plasma after oral administration of Coptidis and coptidis–gardeniae couple extract. The results indicated that the plasma profiles of the two compounds in rabbit confirmed to one-compartment open model and the combinational utilization with Gardeniae could increase the bioavailability of berberine and palmatine, the two major active components of Coptidis.     

Determination of Carvedilol in Human Plasma by High-Performance Liquid Chromatography Applying On-Line Deproteination and Column Switching

An HPLC-column switching method has been developed and validated for the determination of carvedilol in human plasma. 20 μL plasma was injected onto a Brownlee C8 column and the high molecular weight proteins were eluted isocratically. Carvedilol was separated from the majority of the components by a linear acetonitrile gradient. The fraction containing carvedilol was switched on-line onto a LiChrospher RP 18 column and separated isocratically from accompanying compounds applying 0.1 M triethylamine-methanol-acetonitrile = 17: 18:19 (v/v/v). The influence of pH and temperature on retention was investigated on both columns. No interference from other plasma components was observed. Reproducibility of the complete method was better than 4% and the limit of quantitation was 0.8 ng mL^?1.     

Determination of Palmatine in Rabbit Plasma by RP-HPLC with Solid-Phase Extraction

A rapid and specific reversed-phase high performance liquid chromatography (RP-HPLC) method for the determination of palmatine in rabbit plasma has been developed and validated. The chromatographic separation was performed on a C₁₈ column at 40 °C. The mobile phase, delivered at 1.0 mL min^?1, consisted of acetonitrile/phosphate buffer (pH 3.0) 40:60 (v/v). The detection wavelength was set at 345 nm. Palmatine and internal standard (IS) berberine were extracted from plasma by solid-phase extraction using C18 cartridges. Linearity was confirmed in the concentration range of 0.01 to 5 μg mL^?1, the inter-day and intra-day RSDs were within 10.0, the recoveries of palmatine ranged from 93.1 to 110.3, and the limit of detection (LOD, S/N > 3) was 0.002 μg mL^?1. The method is applicable to the determination of palmatine in rabbit plasma after intravenous administration of palmatine.     

An LC–MS–MS Method for the Simultaneous Quantitation of Acyclovir and Valacyclovir in Human Plasma

A simple, sensitive and selective LC–MS–MS method has been developed for the simultaneous determination of acyclovir and valacyclovir in human plasma. Acyclovir and valacyclovir in plasma were concentrated by solid phase extraction and chromatographed on a C18 column using a mobile phase of 0.1% formic acid: methanol (30:70% v/v). The method was validated over a linear range of 47–10,255 and 5–1,075 ng mL^?1 for acyclovir and valacyclovir respectively. The LOQs were 47.6 and 5.0 ng mL^?1. The validated method was applied for the quantitation of acyclovir and valacyclovir from plasma samples in a pharmacokinetic study.     

HPLC with Column Switching Coupled to APCI–MS for Pharmacokinetic Study of Amygdalin in Rabbit Plasma

A fast and validated assay was established for the pharmacokinetic study of amygdalin in Armeniacae Semen in rabbit. The method involved column switching (CS) enrichment, separation, post-column derivatization, and atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Plasma sample was enriched by CS using a MAYI-ODS as the first column. Analytes of interest were isolated and analyzed on a second column of Zorbax SB-C₁₈. To detect amygdalin in plasma samples, a T-piece was connected between the HPLC outlet and the APCI source to add a mixture of dichloromethane and methanol to the eluent by an isocratic pump. Calibration graphs showed good linearity over a range of 1.0–1,280 ng mL⁻¹. The detection limit was 0.2 ng mL⁻¹. The intra- and inter-day accuracies were within 3.9%. The method was successfully applied to a study of the pharmacokinetics of amygdalin after an intravenous injection of amygdalin extracts to rabbits with a dose of 400 mg kg⁻¹. The results indicate that amygdalin is a one-compartment open model with a first order absorption phase.     

Simultaneous determination of losartan and active metabolite EXP3174 in rat plasma by HPLC with column switching

Summary A new liquid chromatographic method with columnswitching has been developed for the simultaneous determination of losartan and its active metabolite, EXP3174 in rat plasma. The plasma samaple was injected onto a precolumn of Lichroprep RP-8 after dilution with 5% acetonitrile in 50 mM phosphoric acid. Polar plasma components were eluted using this diluent. After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by an Inertsil ODS-2 column with acetonitrile-acetate buffer. The method showed excellent precision, accuracy and speed with detection limit 20 ng mL^–1. Total analysis time per sample was less than 40 min and the coefficients of variation for intra and inter-assay were 4.8%. This method has been successfully applied after oral administration of losartan to rat plasma samples.     

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

Summary An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C₁₈ column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min⁻¹. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL⁻¹). The linearity of response was good (r ²≥0.999) over the concentration range 1–250 ng mL⁻¹.     

Simultaneous Quantitation of Tetrahydropalmatine and Protopine in Rabbit Plasma by HPLC–PAD, and Application to Pharmacokinetic Studies

A reversed-phase high-performance liquid chromatographic method coupled with photodiode-array detection (HPLC–PAD) has been developed and validated for simultaneous quantitation of dl-tetrahydropalmatine (dl-THP) and protopine (PTP), two active constituents of the herbal medicine Rhizoma Corydalis, in rabbit plasma. The pharmacokinetics of dl-THP and PTP were researched in rabbits after oral administration of extracts of Rhizoma Corydalis. Optimum separation was achieved on a reversed-phase column with methanol–phosphate buffer (pH 7.75, 65:35, v/v) as mobile phase. The experimental results showed the intra-day and inter-day precision and accuracy of the method were satisfactory for simultaneous determination of dl-THP and PTP at low, middle, and high concentrations. The lower limits of quantitation were 2.05 ng mL^?1 for dl-THP and 2.10 ng mL^?1 for PTP. Plasma concentration–time data for dl-THP were best fitted by the two-compartment linear pharmacokinetic model whereas data for PTP were best fitted by the one-compartment model.     

A sensitive and specific HPLC-MS method for the determination of sophoridine,sophocarpine and matrine in rabbit plasma

A sensitive and specific method was developed for the determination of sophoridine (SRI), sophocarpine (SC) and matrine (MT) in rabbit plasma by HPLC-MS. After an administration of Kuhuang by injection, blood samples were collected and extracted with methanol. The extract solutions were analysed by HPLC-MS method. The separation was performed on a ZORBAX Extend-C18 column using methanol/water/diethylamine (50:50:0.07, v/v/v) as mobile phase. The quinolizidine alkaloids were detected by using mass spectrometry in the SIM mode. There was a good linear relationship between peak area and concentration of analytes over the concentration range of 13.2–995.0 ng mL^–1 for SRI, 7.0–530.0 ng mL^–1 for SC and 8.8–655.0 ng mL^–1 for MT, respectively. The absolute recovery of this method was more than 57% for SRI, 87% for SC and 91% for MT. The accuracy of assay was more than 90%. The limits of detection (LODs) were 6.8 ng mL^–1 for SRI, 3.5 ng mL^–1 for SC and 4.2 ng mL^–1 for MT, respectively. The limits of quantitation (LOQs) were 13.2 ng mL^–1 for SRI, 7.0 ng mL^–1 for SC and 8.8 ng mL^–1 for MT, respectively. The intra-day and inter-day coefficients of variation (RSDs) were less than 10.1, 6.3 and 5.8% for SRI, SC and MT, respectively. The developed method was applied to determine the concentration–time profiles of SRI, SC and MT in rabbit plasma after injection of Kuhuang.     

A Selective LC Method for the Determination of Reboxetine in Human Plasma with Fluorescence Detection

A rapid, simple, accurate, sensitive and reproducible high performance liquid chromatographic method for the quantitation of reboxetine (REB) in human plasma using fluvoxamine as an internal standard (IS) has been developed and validated. The method is based on derivatization with 7-chloro-4-nitrobenzofurazan (NBD-Cl). The NBD-derivatives in plasma were extracted by liquid–liquid extraction and chromatographed on a reversed phase C₁₈ column with isocratic elution using acetonitrile and aqueous nitric acid (pH 3) solution. Calibration curve was linear over the range 2.0–200.0 ng mL⁻¹ with inter- and intra-assay precision (RSD%) of less than 4%. The mean recovery was about 94% for REB. The applicability of the method to the plasma was also studied.

     

LC Determination of Isosorbide-5-Mononitrate in Human Plasma

A simple, sensitive, selective and cost effective LC–UV method was developed for determination of isosorbide mononitrate in human plasma using guaifenesin as an internal standard. Isosorbide mononitrate in plasma was extracted by a single step liquid extraction using tert-butyl methyl ether and chromatographed on a C18 column using water and acetonitrile (80:20 v/v) as mobile phase. The method was validated and exhibited a linear range from 51.6 to 2064.4 ng mL^?1. The inter- and intra-assay accuracy ranged from 97.2–102.7 to 94.2–105.5%, respectively, with precision less than 10% in both the cases. The LLQ was 51.6 ng mL^?1. The validated method was applied to the quantitation of isosorbide mononitrate from plasma samples in a pharmacokinetic study.     

Quantitative Determination of Lobeline Hydrochloride in Rabbit Plasma by LC–MS–MS and Its Application

A sensitive and selective liquid chromatography tandem mass spectrometry method for quantitative determination of lobeline hydrochloride in rabbit plasma was developed and validated. After addition of triazolam as internal standard, protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 column with acetonitrile-0.1% formic acid as mobile phase with gradient elution. Electrospray ionization source was applied and operated in positive ion mode; multiple reaction monitoring mode was used for quantification using target fragment ions m/z 338.1 → 315.8 for lobeline hydrochloride and m/z 342.9 → 308.0 for the IS. Calibration plots were linear over the range of 2–500 ng mL^?1 for lobeline hydrochloride in plasma. Lower limit of quantitation for lobeline hydrochloride was 2 ng mL^?1. Mean recovery of lobeline hydrochloride from plasma was in the range 97.5–102.3%. RSD of intra-day and inter-day precision were both <9%. This developed method is successfully used in pharmacokinetic study of lobeline hydrochloride in rabbit.     

Rapid LC-APCI-MS-MS Method for Simultaneous Determination of Phenacetin and Its Metabolite Paracetamol in Rabbit Plasma

A highly sensitive liquid chromatographic-atmospheric pressure chemical ionization-tandem mass spectrometric method is developed to quantitate phenacetin and its metabolite paracetamol in rabbit plasma. The analytes and internal standard oxazepam are extracted from plasma by liquid–liquid extraction using ethyl acetate, and separated on a Zorbax SB-C₁₈ column (2.1 mm × 150 mm, 5 μm) using acetonitrile–0.1% formic acid in water (40:60 v/v) at a flow of 0.4 mL min^?1. Detection is carried out by multiple reaction monitoring on a ion-trap LC-MS-MS system with an atmospheric pressure chemical ionization interface. The assay is linear over the range 4–1,600 ng mL^?1 for phenacetin and 3–2,000 ng mL^?1 for paracetamol, with a lower limit of quantitation of 4 ng mL^?1 for phenacetin and 3 ng mL^?1 for paracetamol. Intra- and inter-day precision are less than 7.1% and the accuracy are in the range 97.3–103.5%. The validated method is successfully used to analyze the drug in samples of rabbit plasma for pharmacokinetic study.     

Simple and Sensitive High-Performance Liquid Chromatographic Method for Determination of Transdermally Applied Flurbiprofen in Rat Plasma and Excised Skin Samples

A simple reversed-phase HPLC method has been developed for determination of flurbiprofen in rat plasma, excised skin extract, and transdermal patch formulations. The mobile phase was methanol–1% (v/v) phosphoric acid in water, 80:20 (v/v), at a flow rate of 0.5 mL min^-1; ibuprofen was used as internal standard. Flurbiprofen and ibuprofen was detected by UV absorption at 254 nm and 220 nm, respectively. The limit of quantitation was 0.1 µg mL^-1. The response was linearly dependent on concentration in the range 0.1–10 µg mL^-1, and accuracy and reproducibility were good. At these concentrations intraday and interday assay variability were below 8%. Recovery of flurbiprofen was greater than 94% over the linear range of calibration plot.     

Simultaneous LC Determination of Quercetin, Kaempferol and Isorhamnetin in Rabbits after Intragastric Administration of an Ethanol Extract from Pollen Typhae

A simple and rapid reversed-phase LC method was developed and validated for simultaneous determination of three flavonoids, quercetin (QU), kaempferol (KA) and isorhamnetin (IS), in rabbit blood plasma. The plasma was deproteinized using 10% trichloroacetic acid and extracted by n-butanol–acetoacetate solvent prior to LC analysis. The analyte was separated on a reversed-phase column with acetonitrile and 0.1% phosphoric acid in water (27:73, v/v) as mobile phase at a flow-rate of 0.8 mL min^?1, and UV detection wavelength at 369 nm. By this developed method, the concentrations of QU, KA and IS were linearly related to their responses in the range of 0.05–2.5 μg mL^?1. The precision and accuracy for QU, KA and IS in plasma were within ±15% except for the limit of quantitation (LOQ), where they were within ±20%. The validated method has been successfully applied in the pharmacokinetic study of QU, KA and IS in rabbits after intragastric administration of an ethanol extract from traditional Chinese medicine Pollen Typhae.     

Development and Validation of an Stability Indicating RP-LC Method for Determination of Imatinib Mesylate

A new, sensitive and stability indicating liquid chromatographic method has been developed for the determination of imatinib mesylate (IM). Efficient chromatographic separation was achieved using a C₁₈ column with simple mobile phase combination delivered in an isocratic mode and quantitation was carried out using ultraviolet detection. For the first time, a novel microwave assisted degradation procedure was employed for stress testing studies. In addition, orthogonal separation technique was applied to demonstrate selectivity of the proposed method. The method has demonstrated excellent linearity over the range of 25–1,600 ng mL⁻¹. Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL⁻¹) and limit of quantitation (10.16 ng mL⁻¹). The method has shown good and consistent recoveries (99.35–100.69%) with low intra- and inter-day relative standard deviation (RSD) (<2.5%). Experimental design confirmed that peak area was unaffected by small changes in critical factors, in robustness study. The validated method was successfully applied for determination of IM in pharmaceutical formulations.

     

Development and Validation of an Stability Indicating RP-LC Method for Determination of Imatinib Mesylate

A new, sensitive and stability indicating liquid chromatographic method has been developed for the determination of imatinib mesylate (IM). Efficient chromatographic separation was achieved using a C₁₈ column with simple mobile phase combination delivered in an isocratic mode and quantitation was carried out using ultraviolet detection. For the first time, a novel microwave assisted degradation procedure was employed for stress testing studies. In addition, orthogonal separation technique was applied to demonstrate selectivity of the proposed method. The method has demonstrated excellent linearity over the range of 25–1,600 ng mL^?1. Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL^?1) and limit of quantitation (10.16 ng mL^?1). The method has shown good and consistent recoveries (99.35–100.69%) with low intra- and inter-day relative standard deviation (RSD) (<2.5%). Experimental design confirmed that peak area was unaffected by small changes in critical factors, in robustness study. The validated method was successfully applied for determination of IM in pharmaceutical formulations.     

Determination of Gastrodin and Ligustrazine Hydrochloride in Plasma and Brain Dialysate by LC–Tandem MS

A gradient liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of gastrodin and ligustrazine hydrochloride in rat plasma and brain dialysates. Zolpidem was used as internal standard. For plasma samples, solid-phase extraction was used and the brain dialysates were collected from freely moving rats using brain microdialysis. Both were followed by HPLC separation and positive electrospray ionization tandem mass spectrometry detection (ESI–MS–MS). Chromatographic separation was achieved on a Symmetry RP-18 column using gradient elution with methanol and water containing 0.5% formic acid and 2 mM ammonium formate. Selected reaction monitoring (SRM) mode was used for quantitation. Good linearities were obtained in the range of 0.05–100 and 0.01–50 μg mL^?1 for gastrodin and ligustrazine hydrochloride in rat plasma, and 0.05–1,000 ng mL^?1 for both in dialysate. The lower limit of quantitation was 0.01 ng mL^?1 for gastrodin and 0.05 ng mL^?1 for ligustrazine. The method is precise and reliable and can be applied to pharmacokinetic studies.