Liquid Chromatographic Method For Analysis Research Articles

Plasma and Ocular Prednisolone Disposition after Oral Treatment in Cats

Objective. To evaluate the plasma and aqueous humor disposition of prednisolone after oral administration in cats. Methods. Six cats were administered with a single oral dose of prednisolone (10 mg). Blood and aqueous humor samples were serially collected after drug administration. Prednisolone concentrations in plasma and aqueous humor were measured at 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 h after administration by a high-performance liquid chromatographic analytical method developed and validated for this purpose. Results. Mean ± standard error (SE) of maximum plasma prednisolone concentration (300.8 ± 67.3 ng/mL) was reached at 1 h after administration. Prednisolone was distributed to the aqueous humor reaching a mean peak concentration of 100.9 ± 25.5 ng/mL at 1.25 h after administration. The mean ± SE systemic and aqueous humor exposure (AUC) was 553.3 ± 120.0 ng∗h/mL and 378.8 ± 64.9 ng∗h/mL, respectively. A high AUCaqueous humor/AUCplasma ratio was observed (0.68 ± 0.13). The mean half-life time of elimination in plasma and aqueous humor was 0.87 ± 0.16 h and 2.25 ± 0.44 h, respectively. Clinical Significance. The observed high ratio between aqueous humor and plasma prednisolone concentrations indicates that extensive penetration of prednisolone to the anterior segment of the eye may occur. This is the first step that contributes to the optimization of the pharmacological therapeutics for the clinical treatment of uveitis.

Development and validation of a reversed phase liquid chromatographic method for analysis of oxytetracycline and related impurities

A simple, robust and fast high-performance liquid chromatographic method is described for the analysis of oxytetracycline and its related impurities. The principal peak and impurities are all baseline separated in 20min using an Inertsil C8 (150mm×4.6mm, 5μm) column kept at 50°C. The mobile phase consists of a gradient mixture of mobile phases A (0.05% trifluoroacetic acid in water) and B (acetonitrile–methanol–tetrahydrofuran, 80:15:5, v/v/v) pumped at a flow rate of 1.3ml/min. UV detection was performed at 254nm. The developed method was validated for its robustness, sensitivity, precision and linearity in the range from limit of quantification (LOQ) to 120%. The limits of detection (LOD) and LOQ were found to be 0.08μg/ml and 0.32μg/ml, respectively. This method allows the separation of oxytetracycline from all known and 5 unknown impurities, which is better than previously reported in the literature. Moreover, the simple mobile phase composition devoid of non-volatile buffers made the method suitable to interface with mass spectrometry for further characterization of unknown impurities. The developed method has been applied for determination of related substances in oxytetracycline bulk samples available from four manufacturers. The validation results demonstrate that the method is reliable for quantification of oxytetracycline and its impurities.

Analytical method development for directed enzyme evolution research: A high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality l-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a β-cyclobond 2000 analytical column separated ribose and ribitol in 2.3min, a C18 guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4min which provided capability of screening 4×96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25mM tetraborate with 5mM β-cyclodextrin was used as electrolyte. 0.35%of d-ribose in l-ribose can be detected which can be translated into 99.3% ee of l-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.

Determination of tolperisone in human plasma by liquid chromatography/tandem mass spectrometry for clinical application

We have developed and validated a simple, rapid, and sensitive liquid chromatography analytical method employing tandem mass spectrometry (LC–MS/MS) for the determination of tolperisone, a centrally acting muscle relaxant, in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tolperisone was performed using a reversed-phase Luna C18 column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5) – methanol (12:88, v/v) and quantified by tandem mass detection in ESI positive ion mode. The flow rate of the mobile phase was 250μL/min and the retention times of tolperisone and the internal standard (IS, dibucaine) were both 0.6min. The calibration curves were linear over a range of 0.5–300ng/mL (r>0.999). The lower limit of quantification, using 200μL human plasma, was 0.5ng/mL. The mean accuracy and precision for intra- and inter-day validation of tolperisone were within acceptable limits. The LC–MS/MS method reported here showed improved sensitivity for quantification of tolperisone in human plasma compared with previously described analytical methods. Lastly, the validated method was successfully applied to a pharmacokinetic study in humans.

Simultaneous determination of ferulic acid, hesperidin, 6-gingerol and glycyrrhizin in Insampaedok-san by HPLC coupled with diode array detection

For the simultaneous determination of ferulic acid, hesperidin, 6-gingerol and glycyrrhizin in Insampaedok-san, one of traditional oriental medicine prescription, a high performance liquid chromatography (HPLC) analytical method was developed. The separation was achieved on a SHISEIDO C18 column (S-5 μm, 4.6 mm I.D. × 250 mm). The mobile phase consisted of water with 0.1% trifluoroacetic acid and methanol in gradient elution at a flow rate of 1.0 mL/min. The UV wavelength was set at 250, 280 and 320 nm. The method was validated for linearity, precision and recovery. All calibration curves showed good linear regression (r 2 > 0.9994) over the wide test ranges. LOD (limit of detection) and LOQ (limit of qualification) ranged from 0.02 to 0.14 μg/mL and 0.05 to 0.42 μg/mL, respectively. The relative standard deviation (RSD) of the intra- and inter-day test, precision test were within 2.26 and 2.45%, respectively, with accuracy from 95.95 to 108.22%. The recoveries of the four compounds were obtained between 94.30 and 106.34% with RSD less than 1.96%. The verified method was successfully applied to quality control of Insampaedok-san.

Determination of tamsulosin in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

Tamsulosin, a selective α1-adrenoceptor antagonist, is used for the treatment of benign prostatic hyperplasia (BPH). We developed and validated a rapid, sensitive, and simplified liquid chromatography analytical method utilizing tandem mass spectrometry (LC–MS/MS) for the determination of tamsulosin in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tamsulosin was achieved using a reversed-phase Luna C18 column (2.0mm×50mm, 5μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5)–methanol (25:75, v/v) and quantified by MS/MS detection in ESI positive ion mode. The flow rate of the mobile phase was 200μL/min and the retention times of tamsulosin and the internal standard (IS, diphenhydramine) were 0.8 and 0.9min, respectively. The calibration curves were linear over a range of 0.01–20ng/mL (r>0.999). The lower limit of quantification using 500μL of human plasma was 0.01ng/mL. The mean accuracy and precision for intra- and inter-day validation of tamsulosin were both within acceptable limits. The present LC–MS/MS method showed improved sensitivity for quantification of tamsulosin in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.

Development and validation of a reversed-phase liquid chromatographic method for analysis of demeclocycline and related impurities

A simple, robust, and rapid reversedphase high-performance liquid chromatographic method for the analysis of demeclocycline and its impurities is described. Chromatographic separations were achieved on a Symmetry Shield RP8 (75 mm × 4.6 mm, 3.5 μm) column kept at 40°C. The mobile phase was a gradient mixture of acetonitrile, 0.06 M sodium edetate (pH 7.5), 0.06 M tetrapropylammonium hydrogen sulphate (pH 7.5) and water, A (2:35:35:28 v/v/v/v) and B (30:35:35:0 v/v/v/v) pumped at a flow rate of 1 mL/min. UV detection was performed at 280 nm. The developed method was validated according to the ICH guidelines for specificity, limit of detection, limit of quantification, linearity, precision, and robustness. An experimental design was applied for robustness study. Results show that the peak shape, chromatographic resolution between the impurities, and the total analysis time are satisfactory and better than previous methods. The method has been applied for the analysis of commercial demeclocycline bulk samples available on the market.

Determination of Furosemide and Zonisamide as a Drug Substance and in Dosage Form by Ion Pair–Reversed Phase Liquid Chromatographic Technique

An isocratic, selective, and accurate Ion pair - reversed phase liquid chromatographic method of analysis of Furosemide and Zonisamide both as a bulk drug and formulations was developed and validated. An ODS chromatographic column (250mmx4.6mm, 5µm) was used for the separation. The mobile phase consisted of a mixture of methanol and Tetrabutyl ammonium hydrogen sulphate (TBHS) 0.035 molar aqueous solution, pH adjusted to 6.0 using 1 N sodium hydroxide solution. The composition of TBHS with methanol used for Furosemide was 40:60(v/v) and that for Zonisamide was 50:50(v/v) delivered at a flow rate of 1.0ml/min and detection at wave length 240 nm. The developed method was validated in terms of selectivity, Linearity, limit of quantitation, precision, accuracy and solution stability. The proposed LC method achieved satisfactory resolution between Furosemide and 4-Chloro-5-sulphamoyl salicylic acid (CSSA), Zonisamide and CSSA an intermediate product possibly present in Furosemide and Zonisamide. The method can be employed as stability indicating method for both the drugs furosemide and zonisamide and its dosage form.

Quantification of catechins and caffeine from green tea (Camellia sinensis) infusions, extract, and ready-to-drink beverages

This study aimed to quantify the levels of catechins and caffeine in various forms of presentation of green tea: infusion tea bags, extract, and ready-to-drink beverage and, based on their content, identify the most suitable for consumption. High Performance Liquid Chromatography (HPLC) analytical method was used for the quantification of catechins and caffeine. The tea bags had the highest concentration of total catechins with 5 to 9.5% followed by the extract with 3.64 to 4.88%, and ready-to-drink green tea beverage showed low levels of catechins, from 0.14 to 0.26%. As for caffeine content, green tea extract had higher concentration (1.96 to 3.54%) compared to the tea bags (1.39 to 1.57%). Tea bags were found the most suitable for consumption because it contains higher amounts of catechins and smaller amounts of caffeine.

Determination of atomoxetine metabolites in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study

4-Hydroxyatomoxetine (4-HAT) and N-desmethylatomoxetine (N-DAT) are major metabolites of atomoxetine, a potent and selective inhibitor of the presynaptic norepinephrine transporter that is used for the treatment of attention deficit/hyperactivity disorder. The pharmacological activity of 4-HAT is similar to that of atomoxetine. We have developed and validated a simple, rapid and sensitive liquid chromatography analytical method with tandem mass spectrometry (LC–MS/MS) for the determination of 4-HAT and N-DAT in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of analytes was performed using a reversed-phase Luna C18 column (2.0mm×100mm, 3μm particles) with a mobile phase of 10mM ammonium formate buffer (pH 3.5)–methanol (10:90, v/v) and quantified by MS/MS detection in ESI positive ion mode. The flow rate of the mobile phase was 250μL/min and the retention times of 4-HAT, N-DAT and internal standard (IS, metoprolol) were 0.9, 1.0 and 1.0min, respectively. The calibration curves were linear over the range of 0.05–20ng/mL for 4-HAT and 0.1–20ng/mL for N-DAT. The lower limits of quantification, using 200μL human plasma, were 0.05 and 0.1ng/mL for 4-HAT and N-DAT, respectively. The mean accuracy and precision for intra- and inter-day validation of 4-HAT and N-DAT were both within the acceptable limits. This LC–MS/MS method showed improved sensitivity for quantification of the two main metabolites of atomoxetine in human plasma compared with previously described analytical methods. The validated method was successfully applied to a pharmacokinetic study in humans.

Liquid Chromatography Post-Column Oxidation (PCOX) Method for the Determination of Paralytic Shellfish Toxins in Mussels, Clams, Oysters, and Scallops: Collaborative Study

Sixteen laboratories participated in a collaborative study to evaluate method performance parameters of a liquid chromatographic method of analysis for paralytic shellfish toxins (PST) in blue mussels (Mytilus edulis), soft shell clams (Mya arenaria), sea scallops (Placopectin magellanicus), and American oysters (Crassostrea virginicus). The specific analogs tested included saxitoxin, neosaxitoxin, gonyautoxins-1 to -5, decarbamoyl-gonyautoxins-2 and -3, decarbamoyl-saxitoxin, and N-sulfocarbamoyl-gonyautoxin-2 and -3. This instrumental technique has been developed as a replacement for the current AOAC biological method (AOAC Official Method 959.08) and an alternative to the pre-column oxidation LC method (AOAC Official Method 2005.06). The method is based on reversed-phase liquid chromatography with post-column oxidation and fluorescence detection (excitation 330 nm and emission 390 nm). The shellfish samples used in the study were prepared from the edible tissues of clams, mussels, oysters, and scallops to contain concentrations of PST representative of low, medium, and high toxicities and with varying profiles of individual toxins. These concentrations are approximately equivalent to 1/2 maximum level (ML), ML, or 2xML established by regulatory authorities (0.40, 0.80, and 1.60 mg STX diHCl eq/kg, respectively). Recovery for the individual toxins ranged from 104 to 127%, and recovery of total toxin averaged 116%. Horwitz Ratio (HorRat) values for individual toxins in the materials included in the study were generally within the desired range of 0.3 to 2.0. For the estimation of total toxicity in the test materials, the reproducibility relative standard deviation ranged from 4.6 to 20%. A bridging study comparing the results from the study participants using the post-column oxidation (PCOX) method with the results obtained in the study director's laboratory on the same test materials using the accepted reference method, the mouse bioassay (MBA; AOAC Official Method 959.08), showed that the average ratio of results obtained from the two methods was 1.0. A good match of values was also achieved with a new certified reference material. The results from this study demonstrated that the PCOX method is a suitable method of analysis for PST in shellfish tissue and provides both an estimate of total toxicity, equivalent to that determined using the MBAAOAC Official Method 959.08, and a detailed profile of the individual toxin present in the sample.

Validation of an Oil-in-Water Microemulsion Liquid Chromatography Method for Analysis of Perindopril tert-Butylamine and Its Impurities

This paper describes the development and validation of a microemulsion liquid chromatography (MELC) method for simultaneous determination of perindopril tert-butylamine and its impurities in bulk active substances and the pharmaceutical dosage form of tablets. An appropriate resolution with reasonable retention times was obtained for a microemulsion containing 0.24% (w/v) butyl acetate, 0.30% (w/v) ethyl acetate, 2% (w/v) sodium dodecyl sulfate, 7.75% (w/v) n-butanol, and 20.0 mM potassium dihydrogen phosphate, the pH of which was adjusted to 3.70 with 85% orthophosphoric acid. Separations were performed on a Nucleosil 120-5 butyl modified (C4), 250 x 4 mm, 5 microm particle size silica column at 40 degrees C, with a mobile phase flow rate of 1.25 mL/min. UV detection was performed at 254 nm. The established method was subjected to method validation, and required validation parameters were defined. Robustness testing, an important part of method validation, was performed as well. Since robustness validation can be conducted using different experimental designs, the Plackett-Burman design was applied due to its possibility of testing many factors at the same time. The validated MELC method was found to be suitable for the simultaneous determination of perindopril tert-butylamine and its impurities in pharmaceuticals.

SEPARATION, IDENTIFICATION AND QUANTITATION OF DEGRADANTS OF TOPOTECAN AND ITS RELATED IMPURITIES IN TOPOTECAN INJECTION BY RP-HPLC AND LC-MS

A simple, rapid, accurate, robust, sensitive, and stability-indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) analytical method has been developed for the separations of related impurities and degradants of topotecan in topotecan injection. Forced degradation studies performed on topotecan injection formulation revealed that the compound (topotecan) is extremely susceptible to all hydrolytic, oxidation, light exposure, and thermal stress. The method was tested in the presence of commonly used formulation excipients such as mannitol and tartaric acid. Separation of topotecan from its degradation products and three related impurities (Imp-A, Imp-B, Imp-C) as process impurities was achieved on ACE® C18, 150 × 4.6 mm i.d, 3 µm, stainless steel analytical column using a gradient elution. The elution of impurities employed multi-step gradient programmed mobile phase consisting of 0.02 M ammonium acetate (pH, 4.2) as mobile phase-A and a mixture of methanol and isopropyl alcohol in the ratio of 750:250 (v/v), respectively, as mobile phase-B. The mobile phase flow rate was maintained at 0.8 mL min−1. The column eluent was monitored at 267 nm. The column oven temperature was 25°C. The same method was also extended to LC-MS with the same gradient conditions used to identify the degradation products observed under forced degradation studies. The method was validated according to FDA, USP (general chapter 1225), and ICH guidelines, and statistical analysis of the data proved to be suitable for stability testing at quality control. The linearity of the proposed method for all the aforementioned related impurities and topotecan was investigated in the range of LOQ to 4.9 µg mL−1, respectively. The method was shown to separate topotecan well from the degradants and commonly used excipients. The specificity of the method was established through peak purity testing using a photodiode array detector.

Optimization and validation of a new high-performance liquid chromatographic method for analysis of acetazolamide in tablets using Box-Behnken statistical experiment design

Summary This paper describes the optimization and validation of an analytical method for HPLC analysis of acetazolamide in tablets using Box-Behnken design. This multivariate approach enables substantial improvement of chromatographic performance using fewer experiments, without additional cost of columns or other equipment. By use of quadratic regression analysis, equations were developed describing the behavior of the response as simultaneous functions of the selected independent variables. Accordingly, the optimum conditions were determined. For quality-control samples these were use of a C18 column with acetonitrile-phosphoric acid buffer solution (0.01 m, pH 7.1) 15:85 (ν/ν) as mobile phase at a flow rate of 1.0 mL min−1. Diode-array detection was performed at 266 nm. The method was validated, in accordance with ICH guidelines, for accuracy, precision (intra-day and inter-day coefficient of variation <2.0%), selectivity, and linearity (R2 = 0.9993) over the concentration range 10–300 μg mL−1. The met...

Stability-indicating liquid chromatographic method for analysis of pitavastatin calcium in tablet dosage forms

A simple, specific, sensitive, precise, accurate, and robust stability-indicating reversed-phase liquid chromatographic (LC) method has been established for analysis of pitavastatin calcium in tablet dosage forms. LC separation was achieved on a 250 mm × 4.6 mm i.d., 5-μm particle, C18 column with acetonitrile-water-triethylamine 80:19.8:0.2 (υ/υ), adjusted to pH 3.5 ± 0.05 with orthophosphoric acid as isocratic mobile phase at a flow rate of 1.5 mL min−1. Detection, with a photodiode-array detector, was at 238 nm, the wavelength of maximum absorbance in a spectrum obtained from its solution in methanol. The retention time was approximately 5.70 min. The method was validated for linearity, accuracy, precision, limits of detection and quantification, and robustness. Quantification was performed over the concentration range 0.1–2.5 μg mL−1. Mean recovery was 100.26 ± 0.75%. The limit of detection (LOD) was 0.0055 µg mL−1. The method was successfully used for analysis of pitavastatin calcium in tablets and for stability studies, because the method separates pitavastatin calcium from its degradation products and from excipients.

Development and validation of a simple, stability-indicating high-performance liquid chromatographic method for analysis of tolterodine tartrate in the bulk drug and in its tablet formulation

Summary A stability-indicating HPLC method has been developed for analysis of tolterodine tartrate in the bulk drug and in formulations. Acceptable separation of the drug and its degradation products was achieved at 40°C on a 4.6 mm i.d. × 250 mm, 5-μm particle, C18 column with 40:60 (υ/υ) buffer solution-methanol containing 0.5% (υ/υ) triethylamine as mobile phase. The pH of the mobile phase was adjusted to 7.0 ± 0.1 with orthophosphoric acid. The flow rate was 1.2 mL min−1 and the detection wavelength 220 nm. The method was validated for linearity, precision, accuracy, specificity, and robustness. Response was a linear function of concentration over the range 1–100 μg mL−1. The slope of the calibration plot was 17.82 mV s−1 ppm−1, the correlation coefficient 0.999, and the relative standard deviation (RSD) 0.23%. Assessment of precision revealed method RSD was low — from 1.59 to 1.88% for intra-day precision and from 0.59 to 1.90% for inter-day precision. Stress degradation studies showed tolterodine ta...

Validated High Performance Liquid Chromatographic Method for Analysis of Fenvalerate Pesticide in Chilies by QuEChERS Extraction Cleanup and High Liquid Chromatography

Problem statement: Contamination associated with pesticide use has increased as well, adversely impacting the environment and causing human health risks through residues on food. Approach: This study reports the extraction of Fenvalerate (FE) in chilies using QuEChERS based on High Performance Liquid Chromatography (HPLC) describes a simple, fast and inexpensive method. The chillie was extracted with acetonitrile Aliquots were cleaned-up using Solid Phase Extraction (dSPE), a primary-secondary amine carbon black. Reversed-phased HPlc system with PDA detection was used for the separation identification and quantification of all these analyses using acetonitrile methanol-potassium dehydrogenate phosphate (50:40:10) as mobile phase. Results: Limit of detection of 0.01 µg mL was obtained. Calibration curves that constructed for the analyzes spiked into samples followed linear relationships with good correlation coefficients (R2>0.9944). In this method was found to be precise, specific and accurate for detection and analysis of Fenvalerate in chilies. Conclusion: QuEChERS methods are convenient, rugged methods that simplify extract cleanup, reduce material costs and improve sample throughout. Here we demonstrate the effectiveness of QuEChERS sample cleanup using a multiresidue analysis of pesticide on chilies.

Biosynthesis of Cylindrospermopsin and 7-Epicylindrospermopsin in Oscillatoria sp. Strain PCC 6506: Identification of the cyr Gene Cluster and Toxin Analysis

Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 microg/mg and 0.29 microg/mg, respectively) and cylindrospermopsin (0.10 microg/mg and 0.11 microg/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 +/- 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA.

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.

Enantioselective Separation and Determination of Carnosine in Rat Plasma by Fluorescence LC for Stereoselective Pharmacokinetic Studies

A sensitive fluorescence liquid chromatographic analytical method was developed for the simultaneous determination of carnosine enantiomers in rat plasma. The method was applied to pharmacokinetic studies. Chiral separation of carnosine enantiomers was achieved by pre-column derivatization with o-phthaldialdehyde and the thiol N-acety-l-cysteine as derivating reagents. They were separated on an ODS column and detected by fluorescence detection (λex = 350 nm, λem = 450 nm). γ-Aminobutyric acid was used as internal standard. The method was linear up to 6,000 ng mL−1 for l-carnosine, 4,000 ng mL−1 for d-carnosine. Low limit of quantitation (LLOQ) was 40 ng mL−1 for each isomer. The relative standard deviations obtained for intra- and inter-day precision were lower than 12% and the recoveries were higher than 75% for both enantiomers. The method was applied to a stereoselective study on the pharmacokinetics of carnosine after oral administration with a single dose (carnosine, 75 mg kg−1 for each isomer) to a rat. The initial data indicated that l-carnosine had a larger value of the highest plasma concentration than d-carnosine (Cmax 5,344 vs. 1,914 ng mL−1), and that of l-carnosine had a lower value of AUC(0−∞) and t1/2(h) (AUC(0−∞) 5,306 vs. 6,321 ng h mL−1, t1/2 1.43 vs. 3.37 h). Our results indicated that the pharmacokinetic of l-carnosine and d-carnosine revealed enantioselective properties significantly.