Column-switching High-performance Liquid Chromatographic Method Research Articles

Determination of Biogenic Amines in Fish Meat and Fermented Foods Using Column-Switching High-Performance Liquid Chromatography with Fluorescence Detection

In this study, we developed a column-switching high-performance liquid chromatography method with fluorescence detection for the simultaneous analysis of biogenic amines (BAs). This method uses an isocratic solution using acetonitrile with water as the mobile phase. Column-switching is achieved by using a switching valve with a set time program to change flow direction. Using this method, seven BAs (tryptamine, 2-phenylethylamine, putrescine, cadaverine, histamine, tyramine, and spermidine) could be separated and impurities eliminated. Using fluorescence detection, BAs could be identified with high sensitivity. We employed this method to determine the BA contents of fish (mackerel, tuna, and cod) and fermented food (a soybean product and cheese). Our results are in line with previous reports, yielded highly reproducible and quantitative results, and enabled the quick and simultaneous analysis of multiple BAs. Therefore, the method developed here may be useful for the continuous analysis of BAs in different samples to evaluate food quality.

Determination of alendronate in low volumes of plasma by column-switching high-performance liquid chromatography method and its application to pharmacokinetic studies in human plasma

A sensitive and simple column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection was developed for the determination of alendronate in human plasma. Alendronate and pamidronate (internal standard, IS) in plasma including Na(2)EDTA were precipitated with sodium hydroxide and calcium chloride after deproteinization using 10% trichloroacetic acid solution. The precipitated alendronate and IS were reconstituted by sodium citrate and citric acid and then derivatized with 9-fluorenylmethyl chloroformate. The resulting solution was injected onto an HPLC system consisting of a pretreatment column and an analytical column, which were connected with a six-port switching valve. The assay was linear in the concentration range of 2-100 ng/mL in 1 mL of plasma with high precision and accuracy, and the limit of detection was 0.5 ng/mL. It was successfully applied to evaluate the pharmacokinetic parameters of alendronate in human volunteers following single oral administration. The mean value of maximum alendronate plasma concentration (C(max)) was 37.69 ng/mL, and the mean time to reach the C(max) (T(max)) was 1.08 h. The area under the plasma concentration-time curve (AUC) and elimination half-life (T(1/2)) were 106.48 ng/mL/h and 1.66 h, respectively.

Pretherapeutic uracil and dihydrouracil levels of colorectal cancer patients are associated with sex and toxic side effects during adjuvant 5‐fluorouracil–based chemotherapy

In Nordic countries, the standard treatment of colorectal cancer (CRC) in the adjuvant setting is bolus 5-fluorouracil (5-FU) plus leucovorin alone or in combination with oxaliplatin. 5-FU competes with the natural occurring pyrimidine uracil (Ura) as a substrate for dihydropyrimidine dehydrogenase (DPD; enzyme commission number 1.3.1.2). Low DPD activity is associated with toxicity during treatment. Pretherapeutic detection of DPD deficiency could prevent severe toxicity otherwise limiting drug administration. Assays showing that DPD deficiency impairs breakdown of Ura to dihydrouracil (UH(2)) seem promising for clinical use. Urine was collected from 56 untreated volunteers and 143 patients with CRC before adjuvant treatment. Ura and UH(2) were analyzed using a column-switching high-performance liquid chromatography method that incorporates reversed-phase and cation-exchange columns. Ura, UH(2), and UH(2)/Ura levels were related to toxicity. Ura and UH(2) in patients were not different from controls. UH(2) was significantly higher in women compared with men. The UH(2)/Ura ratio, however, did not differ according to sex. Low UH(2) and UH(2)/Ura levels were associated with diarrhea in men. Women experiencing thrombocytopenia had significantly higher Ura compared with women with no thrombocytopenia. The UH(2)/Ura ratio correlated negatively with total toxicity score in men (r = -0.39, P = .020). Pretherapeutic Ura and UH(2) levels per se may be related to risk of side effects during adjuvant 5-FU-based treatment, whereas the UH(2)/Ura ratio may not always reveal such a risk. Sex is a strong risk factor for toxicity, showing the importance of evaluating male and female patients separately.

Development and Validation of a Rapid Column-Switching High-Performance Liquid Chromatographic Method for the Determination of Lamotrigine in Human Serum

This study describes a simple and sensitive column-switching high-performance liquid chromatographic method with UV detection for the determination of Lamotrigine in 50 microL of serum. After solid-phase extraction of Lamotrigine on an Oasis HLB extraction precolumn (20 x 3.9 mm; dp: 25 microm), chromatographic separation was achieved at 30 degrees C on a Chromolith RP-18e column (50 mm x 4.6 mm i.d.) using a solution of 20% acetonitrile in 15 mM phosphate buffer (pH 7.0) as the mobile phase, at a flow-rate of 2.0 mL/min. The eluant was detected at 215 nm. The retention time for Lamotrigine was 1.28 min. The total analysis time was ca. 5 min. However, the overlap of sample preparation, analysis, and reconditioning of the precolumn increased the overall sample throughput to one injection every 3 min. The method was validated for system suitability, linearity, precision, accuracy, robustness, and limit of quantitation. The linearity of the calibration lines, expressed by the linear correlation coefficient, was better than 0.9996. Recovery studies achieved from Lamotrigine spiked plasma samples showed values greater than 93%, demonstrating the excellent extraction efficiency of the precolumn. Intra- and inter-day precision were generally acceptable; the coefficient of variation was < 2.3% in all cases. The detection limits for Lamotrigine at a signal-to-noise ratio of 3 was 0.002 microg/mL when a sample volume of 50 microL was injected. However, it was possible to enhance the sensitivity further by injecting larger volumes, up to 200 microL. The method was shown to be robust and the results were within the acceptable range. The method was successfully applied to the determination of Lamotrigine in human serum samples of patients submitted to Lamotrigine therapy.

Determination of losartan, telmisartan, and valsartan by direct injection of human urine into a column-switching liquid chromatographic system with fluorescence detection

Column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of losartan, telmisartan, and valsartan in human urine. Urine samples were diluted on the extraction mobile phase (1:4, v/v) and a volume of 20 μL of this mixture were directly injected onto the HPLC system. The analytes were extracted from the matrix using an on-line solid-phase extraction procedure involving a precolumn packed with 25 μm C 18 alkyl-diol support (ADS), and a solution 2% methanol in 5 mM phosphate buffer (pH 3.8) at a flow-rate of 0.8 mL/min for isolation and preconcentration of losartan, telmisartan, and valsartan. The enriched analytes were back-flushed after, onto the analytical column with a mixture of 5 mM phosphate buffer (pH 3.8)–acetonitrile–methanol (65:20:15, v/v/v) at a flow-rate of 3.0 mL/min and detected by fluorescence at 259 and 399 nm as excitation and emission wavelength respectively. The separation of losartan, telmisartan, and valsartan was achieved on a Chromolith RP-18e monolithic column. The method provides extraction recoveries from spiked urine samples greater than 93%. Intra-day and inter-day precision were generally acceptable; the intra-day-assay C.V. was <3.5 for all compounds and the inter-day-assay C.V. was <3.7%. The estimated calibration range was 0.001–2.5 μg mL −1 with excellent coefficient of determination (>0.9981). The detection limits for losartan, telmisartan, and valsartan at a signal-to-noise ratio of 5:1 were 0.002, 0.0002 and 0.001 μg mL −1 when a sample volume of 20 μL was injected. The proposed method permitted the simultaneous determination of losartan, telmisartan, and valsartan in 8 min, with an adequate precision and sensitivity. However, the overlap of the sample cleanup step with the analysis increases the sampling frequency to 12 samples/h. The developed column-switching method was successfully applied for the determination of these analytes in human urine samples of patients submitted at ARA-IIs therapy.

Use of a column-switching high-performance liquid chromatography method to assess the presence of specific binding of (R)- and (S)-[11C]rolipram and their labeled metabolites to the phosphodiesterase-4 enzyme in rat plasma and tissues

To complement recent studies using the high-affinity (11)C-labeled phosphodiesterase-4 (PDE4) inhibitor (R)-rolipram and the less active enantiomer (S)-[(11)C]rolipram for in vivo quantification of PDE4 levels, we evaluated the presence of radiolabeled metabolites and their potential binding to PDE4 in the rat plasma, brain, heart, pancreas, skeletal muscle and brown adipose tissue. A reverse-phase capture and analytical HPLC column-switch method was used to detect (R)-[(11)C]rolipram, (S)-[(11)C]rolipram and their radiolabeled metabolites in rat plasma and tissue extracts. The relative proportion of PDE4-specific binding of the radiotracers and their labeled metabolites was analyzed following co-injections with a saturating dose of unlabeled (R)-rolipram at 45 min post-tracer injection in tissue extracts. Radiolabeled metabolites were found in the plasma (72-75% of total radioactive signal), and in the heart, skeletal muscle, pancreas and brown adipose tissue (44-52%), but not in the brain. In comparison to polar labeled metabolites, the proportion of unchanged (R)-[(11)C]rolipram was reduced in PDE4-rich organs by co-injection of unlabeled (R)-rolipram. Conversely, no changes were obtained in brown adipose tissue, or with (S)-[(11)C]rolipram, suggesting that radiolabeled metabolites of (R)-[(11)C]rolipram display no specific binding to PDE4. Radiolabeled hydrophilic metabolites are unlikely to compete with (R)-[(11)C]rolipram for PDE4-specific retention. However, due to the high proportion of the radioactive metabolites in the total radioactive signal, any kinetic modeling calculations in the peripheral tissues will need to take into account the presence of labeled metabolites.

Simultaneous Determination of Warfarin Enantiomers and Its Metabolite in Human Plasma by Column-Switching High-Performance Liquid Chromatography With Chiral Separation

A simple and sensitive column-switching high-performance liquid chromatographic method for the simultaneous determination of warfarin enantiomers and their metabolites, 7-hydroxywarfarin enantiomers, in human plasma is described. Warfarin enantiomers, 7-hydroxywarfarin enantiomers, and an internal standard, diclofenac sodium, were extracted from 1 mL of a plasma sample using diethyl ether-chloroform (80:20, v/v). The extract was injected onto column I (TSK precolumn BSA-C8, 5 microm, 10 mm x 4.6 mm inside diameter) for cleanup and column II (Chiralcel OD-RH analytical column, 150 mm x 4.6 mm inside diameter) coupled with a guard column (Chiralcel OD-RH guard column, 10 mm x4.6 mm inside diameter) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (84:16 v/v, pH 2.0) for clean-up and phosphate buffer-acetonitrile (45:55 v/v, pH 2.0) for separation. The peaks were monitored with an ultraviolet detector set at a wavelength of 312 nm, and total time for chromatographic separation was approximately 25 minutes. The validated concentration ranges of this method were 3 to 1000 ng/mL for (R)- and (S)-warfarin and 3 to 200 ng/mL for (R)- and (S)-7-hydroxywarfarin. Intra- and interday coefficients of variation were less than 4.4% and 4.9% for (R)-warfarin and 4.8% and 4.0% for (S)-warfarin, and 5.1% and 4.2% for (R)-7-hydroxywarfarin and 5.8% and 5.0% for (S)-7-hydroxywarfarin at the different concentrations. The limit of quantification was 3 ng/mL for both warfarin and 7-hydroxywarfarin enantiomers. This method was suitable for therapeutic drug monitoring of warfarin enantiomers and was applied in a pharmacokinetic study requiring the simultaneous determination of warfarin enantiomers and its metabolite, 7-hydroxywarfarin enantiomers, in human volunteers.

Sensitive Determination of Itraconazole and Its Active Metabolite in Human Plasma by Column-switching High-performance Liquid Chromatography With Ultraviolet Detection

A simple and sensitive column-switching high-performance liquid chromatographic method for the simultaneous determination of itraconazole (ITZ) and its active metabolite, hydroxyitraconazole (HIT) in human plasma is described. ITZ, HIT, and an internal standard, R051012, were extracted from 1 mL of alkalinized plasma sample using n-heptane-chloroform (60:40, vol/vol). The extract was injected onto column I (TSK precolumn BSA-ODS/S, 5 microm, 10 x 4.6 mm ID) for clean-up and column II (Develosil C8-5 column, 5 microm, 150 x 4.6 mm ID) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (68:32 vol/vol, pH 6.0) for clean-up and phosphate buffer-acetonitrile (35:65 vol/vol, pH 6.0) for separation. The peaks were monitored with an ultraviolet detector set at a wavelength of 263 nm, and total time for chromatographic separation was about 24 minutes. The validated concentration ranges of this method were 3 to 500 ng/mL for ITZ and 3 to 1000 ng/mL for HIT. Mean recoveries were 59.7% for ITZ and 72.8% for HIT. Intraday and interday coefficients of variation were less than 4.6% and 5.0% for ITZ, and 4.6% and 4.9% for HIT at the different concentrations. The limit of quantification was 3 ng/mL for both ITZ and HIT. This method was suitable for therapeutic drug monitoring of ITZ and HIT, and was applied to pharmacokinetic studies in human volunteers.

Column-switching high-performance liquid chromatographic analysis of fluvastatin in rat plasma by direct injection

A column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of fluvastatin in rat plasma. Plasma samples were diluted with an equal volume of mobile phase, i.e. acetonitrile–5 mM potassium phosphate buffer (pH 6.8) (15:85, v/v), and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then back-flushed onto an analytical column and quantified with fluorescence detection ( λ ex = 305 nm; λ em = 390 nm). The standard curve for the drug was linear in the range 0.5–100 ng mL −1 in rat plasma. The limit of quantitation for plasma was found to be 0.5 ng mL −1. This method has been fully validated and shown to be specific, accurate and precise. The method is simple and rapid because of a minimized sample preparation and appears to be useful for the pharmacokinetic study of fluvastatin.

Sensitive determination of omeprazole and its two main metabolites in human plasma by column-switching high-performance liquid chromatography: Application to pharmacokinetic study in relation to CYP2C19 genotypes

A simple and sensitive column-switching high-performance liquid chromatographic method was developed for the simultaneous determination of omeprazole and its two main metabolites, 5-hydroxyomeprazole and omeprazole sulfone, in human plasma. Omeprazole, its two metabolites and lansoprazol as an internal standard were extracted from 1 ml of alkalinized plasma sample using diethyl ether-dichloromethane (45:55, v/v). The extract was injected into a column I (TSK-PW precolumn, 10 microm, 35 mm x 4.6 mm i.d.) for clean-up and column II (Inertsil ODS-80A column, 5 microm, 150 mm x 4.6mm i.d.) for separation. The mobile phase consisted of phosphate buffer-acetonitrile (92:8 v/v, pH 7.0) for clean-up and phosphate buffer-acetonitrile-methanol (65:30:5 v/v/v, pH 6.5) for separation, respectively. The peak was detected with an ultraviolet detector set at a wavelength of 302 nm, and total time for chromatographic separation was approximately 25 min. The validated concentration ranges of this method were 3-2000 ng/ml for omeprazole, 3-50 ng/ml for 5-hydroxyomeprazole and 3-1000 ng/ml for omeprazole sulfone. Mean recoveries were 84.3% for omeprazole, 64.3% for 5-hydroxyomeprazole and 86.1% for omeprazole sulfone. Intra- and inter-day coefficient variations were less than 5.1 and 6.6% for omeprazole, 4.6 and 5.0% for 5-hydroxyomeprazole and 4.6 and 4.9% for omeprazole sulfone at the different concentrations. The limits of quantification were 3 ng/ml for omeprazole and its metabolites. This method was suitable for use in pharmacokinetic studies in human volunteers, and provides a useful tool for measuring CYP2C19 activity.

Sensitive column-switching high-performance liquid chromatography method for determination of propiverine in human plasma

A sensitive column-switching high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of propiverine in human plasma. Propiverine and internal standard, oxybutynin, were extracted from human plasma that had been made basic with 5N sodium hydroxide into methyl tert-butyl ether. The extracted plasma sample was injected onto the HPLC system consisting of a pretreatment column, a concentrating column, and an analytical column, which were connected with a six-port switching valve. The assay was linear in concentration ranges of 2-200 ng/ml for propiverine in human plasma. This method showed excellent sensitivity (a limit of detection of 0.5 ng/ml), good precision and accuracy. This method is suitable for bioequivalence studies following single dose in healthy volunteers.

Column-switching high-performance liquid chromatographic method for the determination of zaltoprofen in rat plasma

A direct injection column-switching high-performance liquid chromatography (HPLC) method was developed and validated for quantification of zaltoprofen in rat plasma. Following dilution with mobile phase A, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (12:88, v/v) samples were directly injected to the pre-column without sample pre-purification step. After endogenous plasma components were eluted to waste, the system was switched and the analyte was eluted to the trap column. Zaltoprofen was then back-flushed to the analytical column for separation with mobile phase B, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (35:65, v/v) and quantification with an ultraviolet detector at 230 nm. The calibration curve was linear in the concentration range of 40-5000 ngmL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple, rapid and the sample preparation is minimal and appears to be useful for the pharmacokinetic study of zaltoprofen.

Determination of rabeprazole and its active metabolite, rabeprazole thioether in human plasma by column-switching high-performance liquid chromatography and its application to pharmacokinetic study

A new sensitive column-switching high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the simultaneous determination of rabeprazole, a proton pump inhibitor, and its active metabolite, rabeprazole thioether in human plasma. Rabeprazole, its thioether metabolite and 5-methyl-2-[(4-(3-methoxypropoxy)-3-methyl pyridin-2-yl) methyl sulfinyl]-1H-benzimidazole, as an internal standard were extracted from 1 ml of plasma using diethyl ether-dichloromethane (9:1, v/v) mixture and the extract was injected into a column I (TSK-PW precolumn, 10 microm, 35 mmx4.6mm I.D.) for clean-up and column II (C18 Grand ODS-80TM TS analytical column, 5 microm, 250 mmx4.6 mm I.D.) for separation. The peak was detected with an ultraviolet detector set at a wavelength of 288 nm, and the total time for chromatographic separation was approximately 25 min. Mean absolute recoveries were 78.0 and 88.3% for rabeprazole and rabeprazole thioether, respectively. Intra- and inter-day coefficient variations were less than 6.5 and 4.5% for rabeprazole, 3.6 and 5.3% for rabeprazole thioether, respectively, at the different concentration ranges. The validated concentration ranges of this method were 1-1000 ng/ml for rabeprazole and 3-500 ng/ml for rabeprazole thioether. The limits of quantification were 1 ng/ml for rabeprazole and 3 ng/ml for rabeprazole thioether. The method was suitable for therapeutic drug monitoring and was applied to pharmacokinetic study in human volunteers.

Determination of lansoprazole and two of its metabolites by liquid–liquid extraction and automated column-switching high-performance liquid chromatography: application to measuring CYP2C19 activity

A simple and sensitive column-switching high-performance liquid chromatographic (HPLC) method for the simultaneous determination of lansoprazole, a proton pump inhibitor and its major metabolites: 5-hydroxylansoprazole and lansoprazole sulfone in human plasma. The test compounds were extracted from 1 mL of plasma using diethyl ether–dichloromethane (7:3, v/v) mixture and the extract was injected into a column I (TSK-PW precolumn, 10 μm, 3.5 mm × 4.6 mm i.d.) for clean-up and column I (C 18 STR ODS-II analytical column, 5 μm, 150 mm × 4.6 mm i.d.) for separation. The peak was detected by a ultraviolet detector set at a wavelength of 285 nm, and the total time for a chromatographic separation was ∼25 min. The method was validated for the concentration range from 3 to 5000 ng/mL. Mean recoveries were 74.0% for lansoprazole, 68.3% for 5-hydroxylansoprazole, and 79.4% for lansoprazole sulfone. Intra- and inter-day relative standard derivatives were less than 6.1 and 5.1% for lansoprazole, 5.8 and 5.8% for 5-hydroxylansoprazole, 4.4 and 5.9% for lansoprazole sulfone, respectively, at the different concentration ranges. This method is suitable for use in therapeutic drug monitoring and pharmacokinetic studies, and provides use tool for measuring CYP2C19 activity.

Effects of Concomitant Fluvoxamine on the Plasma Concentration of Etizolam in Japanese Psychiatric Patients

Administration of fluvoxamine with concomitant benzodiazepines is common in clinical situations. This study investigated the effects of the coadministration of fluvoxamine on plasma concentrations of etizolam and evaluated the effects of various fluvoxamine doses on drug interactions with etizolam. Subjects were 18 Japanese outpatients concomitantly treated with fluvoxamine before or after monotherapy with etizolam. Plasma concentrations of etizolam were measured using a column-switching high-performance liquid chromatographic method with ultraviolet detection. In 17 subjects treated concomitantly with fluvoxamine at 25 mg or 50 mg, the ranges of plasma concentrations of etizolam corrected for the dose increased from 2.0-13.3 (mean 6.3 +/- 3.6, n = 17) in monotherapy to 2.7-18.2 (mean 9.6 +/- 5.1, n = 17) ng/mL/mg in concomitant doses. Wide variations were observed in the drug interactions; however, coadministration with fluvoxamine produced significant changes in the plasma concentrations of etizolam (P < 0.0001) with a median of 42.9% (range 0.0 to 235.0%). Although the sleepiness of the subjects was evaluated using the Stanford Sleepiness Scale, no changes in sleepiness were found between the etizolam-monotherapy and the fluvoxamine-concomitant states. Of the 12 subjects treated concomitantly with fluvoxamine at 25 mg, 2 subjects received fluvoxamine at a dose increased up to 150 mg, and another received fluvoxamine at a dose increased up to 200 mg. They showed an increase in the plasma concentrations of etizolam in a fluvoxamine dose-dependent manner; more particularly, the increased dose of fluvoxamine (150 mg and 200 mg) resulted in about a twofold variation in plasma concentrations of etizolam.

Simple determination of terbutaline in dog plasma by column-switching liquid chromatography

Terbutaline is a β-adrenergic receptor antagonist that acts as a bronchodilator in the treatment of asthma and chronic bronchitis. In the present work, a column-switching high-performance liquid chromatographic method was developed to monitor terbutaline sulphate in dog plasma. The system consists of a C 2 pre-column (PC) and a C 18 analytical column connected in series via a switching valve. Atenolol was used as the internal standard. Good linearity was achieved in the range of 5–800 ng/ml plasma. The mean intra- and inter-assay variation coefficients for this analysis were 2.3 and 4.7%, respectively. The average recovery for terbutaline was 87.4% from plasma. The mean concentration after three freeze-thaw cycles was 99.4% of the normal value. The analytical sensitivity and accuracy of this assay is adequate for characterisation of the pharmacokinetics of oral administration of terbutaline to dogs and has been successfully used to provide pharmacokinetic data using pulsatile and immediate-release tablets.

Determination of a new atypical antipsychotic agent perospirone and its metabolite in human plasma by automated column-switching high-performance liquid chromatography

A simple and sensitive column-switching high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the quantification of perospirone, a serotonin and dopamine antagonist, and its metabolite ID-15036 in human plasma. The test compounds were extracted from 2 ml of plasma using chloroform–hexane (30:70, v/v) and the extract was injected into a column I (TSK-PW precolumn, 10 μm, 35×4.6 mm I.D.) for clean-up and column II (C 18 STR ODS-II analytical column, 5 μm, 150×4.6 mm I.D.) for separation. The peak was detected using a fluorescence detector set at Ex 315 nm and Em 405 nm, and the total time for a chromatographic separation was ∼30 min. The method was validated for the concentration range from 0.1 to 100 ng/ml. Mean recoveries were 97% for perospirone and 96% for ID-15036. Intra- and inter-day relative standard deviations were less than 2.8 and 5.3% for perospirone and 2.4 and 4.4% for ID-15036, respectively, at the concentration range from 0.3 to 30 ng/ml. This method shows good specificity with respect to commonly prescribed psychotropic drugs, and it could be successfully applied for pharmacokinetic studies and therapeutic drug monitoring.

Direct determination of benzamides in serum by column-switching high-performance liquid chromatography.

A column-switching high-performance liquid chromatographic method with fluorescence detection was developed for the simultaneous determination of four benzamide-type anti-psychotic drugs: sulpiride, tiapride, sultopride and metoclopramide in human serum. In this method, a TSKgel Super-ODS column was used as an analytical column, and a TSKgel G 2000SW was prepared as a pretreatment column. Under the optimized analytical conditions, four benzamide-type anti-psychotic drugs were eluted within 18 min. The detection limits (S/N = 3) for sulpiride, tiapride, sultopride and metoclopramide are 1 ng/ml, 4 ng/ml, 2 ng/ml and 0.5 ng/ml, respectively. Finally, the method was applied to the determination of sulpiride in human serum samples obtained after a single oral dose of sulpiride.

High-performance liquid chromatographic determination of cocaine and benzoylecgonine by direct injection of human blood plasma sample into an alkyl-diol-silica (ADS) precolumn.

A column-switching high-performance liquid chromatographic method with UV detection for the determination of cocaine (COC) and benzoylecgonine (BZE) in human blood plasma samples is described. The method uses an alkyl-diol-silica ADS-C18 extraction precolumn. A 50- micro L plasma sample was introduced to the ADS precolumn in order to separate the analytes from proteins and endogenous compounds. The fraction containing COC and BZE was back-flushed and transferred to an Alltech mixed-mode C(18)/cation-exchange analytical column for final separation. The validation of the method revealed quantitative recoveries from 95.0 to 99.0% for COC at three different concentrations (0.5, 1.0 and 2.0 micro g mL(-1)), and from 96.0 to 99.0% for BZE at the same concentration levels with coefficients of variation <4.00% (n=5). The detection limit (signal to noise ratio (S/N)>3) was 0.03 micro g mL(-1) for all the compounds with an injection volume of 50 micro L. However, it was possible to enhance the sensitivity further by injecting larger plasma volumes, up to 200 micro L, at the same optimal conditions. The overlap of sample preparation, analysis and reconditioning of the extraction column, increase the overall sample throughput to 5 samples h(-1). The developed method has been applied to human blood plasma samples from subjects suspected of cocaine abuse.

Determination of d- and l-aspartate in cell culturing medium, within cells of MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatographic method

HPLC fluorometric methods have been used to analyze trace amounts of d-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis of d-amino acids, in particular d-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for d-Asp in culturing medium is 5 n M. The resulting peak heights correlated well with concentrations that ranged from 12.5 to 250 n M for both d- and l-Asp. The present method was applied to determine d- and l-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels of d- and l-Asp agree with those detected by our previous method. In addition, this method was used to measure d- and l-Asp levels in rat blood samples, and the results are consistent with the reported values.