On-line sample cleanup and enrichment chromatographic technique for the determination of ambroxol in human serum

A sensitive and efficient on-line clean up and pre-concentration method has been developed using column-switching technique and protein-coated μ-Bondapak CN silica pre-column for quantification of ambroxol (AM) in human serum. The method is performed by direct injection of serum sample onto a protein-coated μ-Bondapak CN silica pre-column, where AM is pre-concentrated and retained, while proteins and very polar constituents are washed to waste using a phosphate buffer saline (pH 7.4). The retained analyte on the pre-column is directed onto a C(18) analytical column for separation, with a mobile phase consisting of a mixture of methanol and distilled deionized water (containing 1% triethylamine adjusted to pH 3.5 with ortho-phosphoric acid) in the ratio of 50:50 (v/v). Detection is performed at 254 nm. The calibration curve is linear over the concentration range of 12-120 ng/mL (r(2) = 0.9995). The recovery, selectivity, linearity, precision, and accuracy of the method are convenient for pharmacokinetic studies or routine assays.     

Surface modification by calcium ions—Effect on chromatographic properties of silica

Summary A liquid chromatographic method incorporating column-switching and fluorimetric detection for the determination of triamterene in untreated urine, is described. The urine samples (5 L) were directly introduced onto an Hypersil ODS-C18, 30 m (20 mm×2.1 mm I.D.) pre-column. Polar urinary compounds were removed by flushing the pre-column with water for 1 min, and the analyte was then switched onto an HP-LiChrospher RP C18,5 m (125 mm×4mm ID) analytical column using an acetonitrile/phosphate buffer gradient elution. Fluorescence detection was performed at 230 nm excitation and 430 nm emission wavelengths. The recovery of drug was 102±2% in the 0.10–20.0 g/mL concentration range, the limit of detection being 5 ng/mL. A validation of the usefulness of this procedure was accomplished by analysing urine extracts obtained from real samples.Hypersil ODS is not a product of Merck, Germany. Please give supplier (p. 5).     

使用限进介质色谱柱的柱切换高效液相色谱法直接进样测定大鼠血浆中舒必利

提出一种直接进样测定大鼠血浆中舒必利浓度的高效液相色谱方法,使用限进介质色谱柱作为预柱在线去除血浆蛋白后,将舒必利通过柱切换转移到分析柱中进行分析。限进介质色谱柱为CAPCELLPAKMFSCX阳离子交换柱（20×4．0mmi．d．,5μm）,分析柱为Kromasil C18柱（150×4．6mm i．d．,5μm）,限进介质柱预分离时流动相为PH=6．88的50mmol／L磷酸盐缓冲液乙腈（100：5,V／V）,切换后分析流动相为PH=6．83的50mmol／L磷酸盐缓冲液-乙腈（100：10,V／V）。流速均为1mL／min,检测波长为240nm。该方法检出限为17ng／mL,定量限为50ng／mL。舒必利在50～1400ng／mL之间线性良好（r=0．9997）,高中低浓度的日内、日间相对标准偏差分别为1．5％～4．2％及2．0％～5．2％,方法回收率为98．8％～104．1％．     

Determination of a new non-benzodiazepine anxiolytic and its O-demethyl metabolite in plasma by high-performance liquid chromatography using automated column-switching

An highly sensitive and fully automated high-performance liquid chromatographic assay was developed for the determination of a novel non-benzodiazepine anxiolytic (I) [(R)-2-(methoxymethyl)-1-[(7-oxo-8-phenyl-7H-thieno[2,3-a]quinolizin+ ++- 10-yl)carbonyl]pyrrolidine] and its O-demethyl metabolite (II) in plasma, using column-switching for direct injection of plasma samples. After dilution in internal standard solution, the sample was injected onto a pre-column (17 mm x 4.6 mm) dry-packed with pellicular C18 reversed-phase material. Polar plasma components were removed by flushing the pre-column with water-acetonitrile (90:10, v/v). Retained substances, including I and II, were backflushed onto an analytical column, separated by gradient elution and detected by means of fluorescence detection (excitation, 304 nm; emission, 475 nm). After washing the analytical column and re-equilibrating the pre-column, the system was ready for the next injection. The limit of quantification for I and II was 0.25 and 0.5 ng/ml, respectively, using a 350-microliter specimen of plasma. The practicability of the new method was demonstrated by analysis of more than 300 plasma samples from a tolerance study performed with human volunteers. Owing to its high sensitivity, the method can be used to calculate pharmacokinetic parameters of compounds I and II in man after a single oral dose of about 1 mg of I.     

Rapid, sensitive and fully automated high-performance liquid chromatographic assay with fluorescence detection for sulmazole and metabolites

Sulmazole (2-[(2-methoxy-4-methylsulfinyl)phenyl]-3H-imidazo [4,5-b] pyridine; AR-L 115 BS) and two metabolites (sulfide, sulfone) were quantified from directly injected body fluids (plasma, urine, bile) after high-performance liquid chromatographic separation. No internal standard is needed, which is particularly advantageous when fluorescence detection is established. After automated pre-column enrichment on Corasil C18 (37-50 microns), the parent compound and biotransformation products could be backflushed and chromatographed on ODS-Hypersil (5 microns) with a mixture of 0.075 mol/l phosphate buffer-acetonitrile (2:1), an elution rate of 2.0 ml/min and fluorimetric detection (lambda ex = 330 nm; lambda em = 370 nm). A hydroxylated metabolite of sulmazole which occurs preferentially in urine (and bile) can be quantified in the above-mentioned solvent system diluted 1:1 with water, but with different fluorescence characteristics (lambda ex = 345 nm; lambda em = 515 nm). The assay was linear in the range 8-1000 ng/ml. The lower limit of detection was about 8 ng/ml or 80 pg with coefficients of variation between 0.4 and 5.8% for sulmazole.     

HPLC-F analysis of melatonin and resveratrol isomers in wine using an SPE procedure

An original analytical method has been developed for the determination of the antioxidants trans-resveratrol (t-RSV) and cis-resveratrol (c-RSV) and of melatonin (MLT) in red and white wine. The method is based on HPLC coupled to fluorescence detection. Separation was obtained by using a RP column (C8, 150 mm x 4.6 mm id, 5 mum) and a mobile phase composed of 79% aqueous phosphate buffer at pH 3.0 and 21% ACN. Fluorescence intensity was monitored at lambda = 386 nm while exciting at lambda = 298 nm, mirtazapine was used as the internal standard. A careful pretreatment of wine samples was developed, using SPE with C18 cartridges (100 mg, 1 mL). The calibration curves were linear over the following concentration ranges: 0.03-5.00 ng/mL for MLT, 3-500 ng/mL for t-RSV and 1-150 ng/mL for c-RSV. The LOD values were 0.01 ng/mL for MLT, 1 ng/mL for t-RSV and 0.3 ng/mL for c-RSV. Precision data, as well as extraction yield and sample purification results, were satisfactory. Thus, the method seems to be suitable for the analysis of MLT and resveratrol isomers in wine samples. Moreover, wine total polyphenol content and antioxidant activity were evaluated.     

Automated analysis of fluvoxamine in rat plasma using a column-switching system and ion-pair high-performance liquid chromatography

We have established a robust, fully automated analytical method for the analysis of fluvoxamine in rat plasma using a column-switching ion-pair high-performance chromatography system. The plasma sample was injected onto a precolumn packed with Shim-pack MAYI-ODS (50 microm), where the drug was automatically purified and enriched by on-line solid-phase extraction. After elution of the plasma proteins, the analyte was back-flushed from the precolumn and then separated isocratically on a reversed-phase C18 column (L-column ODS) with a mobile phase (acetonitrile-0.1% phosphoric acid, 36:64, v/v) containing 2 mM sodium 1-octanesulfonate. The analyte was monitored by a UV detector at a wavelength of 254 nm. The calibration line for fluvoxamine showed good linearity in the range of 5-5000 ng/mL (r > 0.999) with the limit of quantification of 5 ng/mL (RSD = 6.51%). Accuracy ranged from -2.94 to 4.82%, and the within- and between-day precision of the assay was better than 8% across the calibration range. The analytical sensitivity and accuracy of this assay is suitable for characterization of the pharmacokinetics of orally-administered fluvoxamine in rats.     

High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenylmethylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-micron, 250 X 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile-water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile-methanol-tetrahydrofuran-water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (lambda max = 264) was about 5 ng/ml and by fluorescence detection (lambda excitation = 270 nm, lambda emission = 300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 +/- 4.2% from serum and 102.0 +/- 2.62% from urine.     

Simultaneous determination of glucose, 1,5-anhydro-d-glucitol and related sugar alcohols in serum by high-performance liquid chromatography with benzoic acid derivatization

A new, simple and sensitive pre-column high-performance chromatographic method for the determination of diabetes marker d-glucose, 1,5-anhydro-d-glucitol and related compounds is reported. Sugars (d-glucose, d-galactose, d-mannose, sucrose and arabinose) were derivatized with benzoic acid (BA) at 80 degrees C for 60 min. l-Fucose, fructose, d-lactose, l-rhamnose, arabinose and ascorbic acid were not reacted. Sugar alcohols (xylitol, erythritol, mannitol, sorbitol myo-inositol) were also derivatized with BA at 80 degrees C for 60 min. The fluorescence derivatives were separated on a TSK amide 80 column (4.6 mm i.d. x 250 mm, 5 microm) with acetonitrile-50 mm acetate buffer (pH 5.6; 4:96, v/v) as the mobile phase. The detection wavelength of beizoic acid derivatives was lambda(ex) 275 nm and lambda(em) 315 nm. The detection limits of sugars were 10-80 microg/mL. The calibration graphs were linear up to 10 mg/mL. The relative standard deviations of 500 microg/mL sugars were 7.0-7.3%. The proposed method was compared with the enzymatic photometric glucose analysis method (Glucose B-Test II Wako). The correlation coefficient was 0.83 (n = 20) and y = 0.82x + 5.91, where y and x are concentrations in microg/mL obtained by the proposed pre-column HPLC and enzyme-photometric method, respectively. The detection limits of sugar alcohols were 100-1000 ng/mL. The calibration graphs were linear to 50 microg/mL and relative standard deviations of 10 microg/mL were 7.2-8.2%. The 1,5-AG data by the proposed method was also compared with the enzymatic photometric 1,5-AG analysis method (Rana AG 1,5-AG determination kit, Nihon Kayaku) and good correlation (r = 0.91, n = 20) was also obtained. The proposed method was applied to the simultaneous determination of d-glucose, 1,5-AG and related sugar alcohols in serum from healthy males.     

Assay of urinary 8‐hydroxy‐2′‐deoxyguanosine by capillary electrophoresis with spectrophotometric detection using a high‐sensitivity detection cell and solid‐phase extraction

A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z‐cell was devised for the assay of 8‐hydroxy‐2′‐deoxyguanosine (8OHdG) in human urine. Solid‐phase extraction (SPE) based on hydrophilic‐lipophilic‐balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused‐silica capillaries employing a Z‐cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10–1000 ng/mL; R² = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.     

Determination of bupropion using liquid chromatography with fluorescence detection in pharmaceutical preparations, human plasma and human urine

A novel pre-column derivatization reversed-phase high-performance liquid chromatography with fluorescence detection is described for the determination of bupropion in pharmaceutical preparation, human plasma and human urine using mexiletine as internal standard. The proposed method is based on the reaction of 4-chloro-7-nitrobenzofurazan (NBD-Cl) with bupropion to produce a fluorescent derivative. The derivative formed is monitored on a C18 (150 mm × 4.6 mm i.d., 5 μm) column using a mobile phase consisting of methanol-water 75:25 (v/v), at a flow-rate of 1.2 mL/min and detected fluorimetrically at λ(ex) = 458 and λ(em) = 533 nm. The assay was linear over the concentration ranges of 5-500 and 10-500 ng/mL for plasma and urine, respectively. The limits of detection and quantification were calculated to be 0.24 and 0.72 ng/mL for plasma and urine, respectively (inter-day results). The recoveries obtained for plasma and urine were 97.12% ± 0.45 and 96.00% ± 0.45, respectively. The method presents good performance in terms of precision, accuracy, specificity, linearity, detection and quantification limits and robustness. The proposed method is applied to determine bupropion in commercially available tablets. The results were compared with an ultraviolet spectrophotometry method using t- and F-tests.     

High-performance liquid chromatographic assay of fleroxacin in human serum using fluorescence detection

A HPLC method has been developed for the direct assay of fleroxacin in serum, without previous extraction. Serum samples, after the addition of sodium dodecylsulfate (0.5%), were injected directly into an LC Hisep column. The mobile phase consisted of acetonitrile, water and triethylamine in a per cent volume ratio 18:80:2. The pH of the mobile phase was adjusted to 6.50 with the addition of phosphoric acid. The drug was detected fluorometrically at lambda (ex )=280 nm and lambda (em )=450 nm . The linear concentration range of fleroxacin was between 0.01 and 2.0mg/l with a detection limit of 1ng/ml.     

Simple determination of azasetron in rat plasma by column-switching high-performance liquid chromatography

For the quantification of azasetron in rat plasma samples, a column-switching HPLC method was developed and validated. Following dilution of plasma samples with mobile phase A (17?mM potassium phosphate buffer (pH 3.0)) and simple protein precipitation by addition of perchloric acid (60%), the mixture was directly injected onto the pre-column. After endogenous plasma substances were eluted to waste, the analyte was transferred to the trap column by switching the system. Then, the analyte was back-flushed to the analytical column for separation with mobile phase B (a 22:78 v/v mixture of acetonitrile and 17?mM potassium phosphate buffer (pH 3.0)) and detected at 250?nm using a photodiode array detector. A linear standard curve was obtained in the concentration range of 10-800?ng/mL with the correlation coefficient (r) of 0.9998. The intra- and inter-day precision and accuracy values for azasetron were in the ranges of 0.3-12.9% and 89.7-101.4%, respectively. The method was valid in terms of specificity, precision, and accuracy. In addition, this efficient analytical method was successfully applied to determine plasma concentrations of azasetron following oral administration of azasetron at a dose of 4.0?mg/kg to rats.     

Liquid chromatographic determination of enrofloxacin in nasal secretions and plasma of healthy pigs using restricted access material for on-line sample clean-up

A new fully automated method was developed for the quantitative analysis of an antibacterial drug, enrofloxacin (ENRO), in both nasal secretions and plasma samples of healthy pigs. The method is based on the use of a pre-column packed with restricted access material (RAM), namely RP-18 ADS (alkyl diol silica), for on-line sample clean-up coupled to a liquid chromatographic (LC) column containing octadecyl silica. The only off-line sample preparation was the 50-fold dilution of nasal secretions and plasma samples in the washing liquid composed of 25 mM phosphate buffer of pH 7.4. A 10 microl diluted sample volume was injected directly onto the pre-column and washed for 7 min. By rotation of a switching valve, the analyte of interest was eluted in the back-flush mode with the LC mobile phase which consisted in a mixture of 25 mM phosphate buffer of pH 3.0 and acetonitrile according to a segmented gradient elution. By a new rotation of the switching valve, the pre-column and the analytical column were equilibrated for 3 min with the initial mobile phases. The flow-rate was 0.8 ml min(-1) for the washing liquid and 1.5 ml min(-1) for the LC mobile phase. ENRO was detected by fluorescence at excitation and emission wavelengths of 278 and 445 nm, respectively. Finally, the developed method was validated using an original strategy based on total measurement error and accuracy profiles as a decision tool. The limits of quantitation of ENRO in plasma and in nasal secretions were 30.5 and 91.6 ng/ml, respectively. The validated method was then applied successfully to the determination of ENRO in healthy pigs treated by intramuscular injection at different doses (2.5, 10 and 30 mg/kg bodyweight) for a pilot study. This method could be also used for the simultaneous analysis of ENRO and its main metabolite, ciprofloxacin (CIPRO).     

High-throughput liquid-chromatography method with fluorescence detection for reciprocal determination of furosemide or norfloxacin in human plasma

A simple, high-throughput, highly selective and sensitive HPLC-FLD method for isolation and determination of furosemide and/or norfloxacin in human plasma samples following a simple organic solvent deproteinization step with acetonitrile as sample 'clean-up' procedure is reported. One of the two drug substances plays the internal standard role for the determination of the other. Separation of analyte and internal standard was achieved in less than 5.3 min (injection to injection) on a Chromolith Performance RP-18e column, using an aqueous component containing 0.015 mol/L sodium heptane-sulfonate and 0.2% triethylamine brought to pH = 2.5 with H(3)PO(4). The composition of the mobile phase was: acetonitrile-methanol-aqueous component = 70:15:15 (v/v/v) and the flow-rate was set up to 3 mL/min. The chromatographic method applied to the determination of furosemide relies on fluorescent detection parameters of 235 nm for the excitation wavelength, and 402 nm for the emission wavelength. In case of norfloxacin, the excitation wavelength is set up to 268 nm and the emission wavelength is set up to 445 nm. The overall method leads to quantitation limits of about 27 ng/mL for furosemide, and 19.5 ng/mL for norfloxacin, using an injection volume of 250 microL. The method was applied to the bioequivalence study of two furosemide-containing formulations.     

Characterization of the precolumn bio trap 500 C18 for direct injection of plasma samples in a column-switching system

Summary A new restricted access media (RAM) type of precolumn, Bio Trap 500 C₁₈, for direct injection of plasma samples in column-switching systems was evaluated with respect to the elution of plasma proteins in different mobile phases, the loading capacity of plasma samples, the chromatographic behavior during plasma injections and protein contamination of the packing and sealings. More than 95% of plasma proteins could be excluded from the precolumn within three minutes for all selected mobile phases. Quantitative analyte recoveries could be obtained by injecting plasma samples ranging from 5 to 500 μL with the analyte mass>150 ng onto a BioTrap 500 C₁₈ column (20×4 mm I.D.). One precolumn tolerated about 15 mL of plasma injection without out noticeable change in retention and pressure. Clogging of the precolumn was encountered (≥45 mL of plasma) due mainly to the adsorption of proteins on the packing. The performance of the analytical column (Kromasil C₁₈) was also examined. The column efficiency decreased by 60% after processing 45 mL plasma in total.     

Automated on-line liquid chromatography-photodiode array-mass spectrometry method with dilution line for the determination of bisphenol A and 4-octylphenol in serum

A novel on-line liquid chromatography-photodiode array detection-mass spectrometry (LC-DAD-MS) system was established with restricted-access media (RAM) pre-column and dilution line combined with a column-switching valve. The serum samples were injected directly onto pre-column under diluted condition by dilution line. After elution of proteins in the serum, the analytes were backflushed onto an ODS analytical column using a six-port column-switching device. The influence of the composition of the mobile phase, for instance, organic modifer, ionic strength, pH, dilution times and the rotation time of the switching valve have been investigated using bisphenol A (BPA) and 4-octyphenol (4-OP) as analytes. The evaluations for peak responses and sensitivity were conducted by MS, and proteins were removed by RAM-column with DAD monitoring at 280 nm. The peak shape was improved by adding a dilution line, especially in the case of large volume injection (LVI), which increased the sensitivity of the analysis. The selective and sensitive quantification of BPA and 4-OP in serum sample could be finished within 25 min. The method had linearity in the range 0.1-500 ng/mL with a limit of quantification for BPA and 4-OP of 0.1 and 0.5 ng/mL, respectively. The recoveries were in the range of 80-101% with less than 9.0% RSDs. This on-line LC-MS method demonstrates potential application to evaluating the exposure and risk of BPA and 4-OP in human.     

Liquid chromatographic/mass spectrometric assay of rabeprazole in dog plasma for a pharmacokinetic study

In order to evaluate the pharmacokinetic (PK) profile of rabeprazole (RA) sterile powder for injection, a rapid, sensitive and specific assay for quantitative determination of RA in dog plasma was developed and validated. After a liquid-liquid extraction procedure, samples were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) using omepazole as the internal standard (IS). The analyte and IS was chromatographed on a ZORBAX Extend-C(18) analytical column (50 x 2 mm i.d, 5 microm, Agilent Technologies, USA). The assay was linear in the range 1-2000 ng/mL. The lower limit of quantification of RA was 1 ng/mL. The recovery of RA was greater than 70%. The within- and between-batch accuracy was 102.7-107.4% and 103.5-105.7%, respectively. The plasma samples for the PK study were collected at defined time points during and after an intravenous injection (1 mg/kg) to beagle dogs and analyzed by LC-ESI-MS method. The PK parameters, such as half-life, volume of distribution, total clearance and elimination rate constant, were determined. The PK profile of RA gave insights into the application in the clinics.     

Simultaneous analysis of five antidepressant drugs using direct injection of biofluids in a capillary restricted-access media-liquid chromatography-tandem mass spectrometry system

Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 microm of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 10(3) less sample than using conventional methods).     

Determination of ofloxacin in human serum by high-performance liquid chromatography with column switching.

The chromatographic behaviour of ofloxacin on various sorbents, including ODS, C8, C1, nitril, phenyl and tert,-butyl, as stationary phases was investigated and a high-performance liquid chromatography (HPLC) assay was developed for the determination of ofloxacin in serum. The serum samples were directly introduced onto an HPLC column after filtering through a Morcut II membrane filter to remove proteins. The filtrate was concentrated on a pre-column using a phenyl stationary phase and was then introduced to an analytical column with an ODS stationary phase by column switching. Ofloxacin and enoxacin as an internal standard were detected by ultraviolet absorbance at 300 nm. Determination was possible for ofloxacin over the concentration range 50-2000 ng/ml; the limit of detection was 20 ng/ml. The recovery of ofloxacin added to serum was 88.8-101.7% with a coefficient of variation of less than 5.2%. This method is applicable to pharmacokinetic studies of patients after treatment with ofloxacin.