Simple High-performance Liquid Chromatographic Method Research Articles

Simultaneous HPLC Determination of Four α-Blocker Drugs Using Time Programming Technique

A sensitive and simple reversed-phase high-performance liquid chromatographic method (RP-HPLC) was developed and validated for the simultaneous determination of four α-adrenergic receptor blockers: terazosin (TER), alfuzosin (ALF), tamsulosin (TAM) and doxazosin (DOX) using moxifloxacin (MOX) as an internal standard. The chromatographic separation was carried out isocratically at 40°C on a Prontosil C-18 reversed-phase column with a mobile phase consisting of acetonitrile : 50 mM phosphate buffer in a ratio of 30:70 with pH 3 adjusted by o-phosphoric acid. The analysis was performed in less than 8 min at a flow rate of 1.0 mL/min for terazosin and alfuzosin, and a flow rate of 1.5 mL/min for tamsulosin and doxazosin using time programming technique. UV-detection was performed at 230 nm. The proposed method was linear over concentration ranges of 2.5 - 25.0 μg/mL for TER, ALF, TAM and DOX, with limits of detection of 0.23, 0.17, 0.20 and 0.28 μg/mL in a respective order. The developed method was successfully applied to determine the four drugs in their dosage forms and the results were satisfactory as revealed by statistical analysis of the data.

Development and Validation of a Simple High-Performance Liquid Chromatography Method for Estimation of Felodipine in Rabbit Plasma: Application to Pharmacokinetic Study

Background: A new, simple, specific and sensitive reverse-phase liquid chromatographic method was developed and validated for the estimation of felodipine (FDP) in rabbit plasma using fluorescence detector. Materials and Methods: Plasma standard were prepared using a simple and one-step protein precipitation method and 50 μ l of the standard solutions were injected into the column. The method was found to be linear in the concentration range of 10–1000 ng/ml. The developed bioanalytical method was validated as per standard guidelines. Validation study results demonstrated accuracy, precision, sensitivity and selectivity of the proposed method. Results: The developed method was successfully employed in the determination of FDP in plasma samples obtained from the pharmacokinetic study of the immediate release tablets in rabbits. Keywords: Felodipine, fluorescence detector, high-performance liquid chromatography, rabbit plasma, pharmacokinetic study, oral tablet.

Fingerprint and multicomponent quantitative analysis for the quality evaluation of Sibiraea angustata leaves by HPLC-DAD and HPLC-ESI-MS/MS combined with chemometrics

ABSTRACTSibiraea angustata leaves, known as a traditional Tibetan medicine, have been specially used in the treatment of indigestion and obesity. In the study, a simple and sensitive high-performance liquid chromatography (HPLC) method with a diode array detector (DAD) was established to solve the problem of lacking quality standard of S. angustata leaves, including the fingerprint analysis and quantification of six characteristic components. The analytical method was validated for linearity, repeatability, stability, recovery, and specificity. Seventeen raw samples and 1 processed sample of S. angustata leaves were collected from different locations of China to establish the fingerprint. The chemometric methods, including similarity analysis (SA), principal component analysis (PCA), and hierarchical clustering analysis (HCA), were applied to distinguish the 18 batches of S. angustata samples. The results successfully sorted these samples into five clusters and kept in line with each other. According to the result of the fingerprint analysis, 21 peaks were extracted to be the common peaks and most of them were identified by mass spectrometry (MS) with electron-spray ionization (ESI) in the negative mode. Meanwhile, the loading plot of PCA further indicated that the peaks of neochlorogenic acid, chlorogenic acid, ferulic acid, rutin, hyperin, and isoquercitrin played a greater role in the discrimination among the 21 peaks. So the six components mentioned above were investigated as index constituents to evaluate the quality of S. angustata leaves from different locations. The study demonstrated that the developed new method was a beneficial approach for authentication and quality evaluation of S. angustata leaves.

A simple HPLC method for the determination of butorphanol tartrate and ondansetron hydrochloride in patient-controlled analgesia solution

A simple and rapid high-performance liquid chromatographic (HPLC) method was established for simultaneous determination of butorphanol tartrate and ondansetron hydrochloride in analgesic mixture samples used for patient-controlled analgesia (PCA). The separation of butorphanol tartrate and ondansetron hydrochloride in PCA solution was carried out on phenomenex C18 column (4.6 mm × 150 mm, 5 μm) using 50 mM sodium acetate (pH 4.0) buffer and acetonitrile (72:28, v/v). Flow rate was 1.0 mL min−1 with a column temperature of 30 °C, and detection wavelength was carried out at 280 nm and 306 nm. Validation of the method was made in terms of specificity, linearity, accuracy, and intra- and inter-day precision, as well as quantification and detection limits. The developed method was successfully used to evaluate the chemical stability of butorphanol tartrate and ondansetron hydrochloride in analgesic mixtures at the usual concentration used for PCA.

A Rapid and Simple High-Performance Liquid Chromatographic Method for Determination of Levofloxacin in Human Plasma

A simple and rapid high-performance liquid chromatography method was developed and validated for quantifying LEV in human plasma. Chromatographic separation was performed under isocratic elution on a Luna Phenomenex® C18 (150 × 4.6 mm, 5 µm) column. The mobile phase was comprised of acetonitrile, methanol, and phosphate buffer 25 mM pH 3.0 (13:7:80 v/v/v) and pumped at a flow rate of 1.5 mL/min. Detection was performed by UV detector at a wavelength of 280 nm. Samples were pre-treated with acetonitrile followed by centrifugation, evaporation, and reconstitution step. The method proved linear (r = 0.995), sensitive (LLOQ and LOD was 1.8 and 0.6 µg/mL respectively), accurate (% error above LLOQ ≤ 12% and LLOQ ≤ 20%), precise (RSD ≤ 9%), robust in the ranges of 1.8-28.8 µg/mL, rapid (separation time not more than 10 min), and simple (use no organic additive in mobile phase). The method was showed reliable for quantifying LEV in human plasma.

Validated RP-HPLC/DAD Method for the Quantification of Insect Repellent Ethyl 2-Aminobenzoate in Membrane-Moderated Matrix Type Monolithic Polymeric Device.

A simple, accurate and sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed for the estimation of ethyl 2-aminobenzoate (EAB) in a matrix type monolithic polymeric device and validated as per the International Conference on Harmonization guidelines. The analysis was performed isocratically on a ZORBAX Eclipse plus C18 analytical column (250 × 4.4 mm, 5 μm) and a diode array detector (DAD) using acetonitrile and water (75:25 v/v) as the mobile phase by keeping the flow-rate constant at 1.0 mL/min. Determination of EAB was not interfered in the presence of excipients. Inter- and intra-day relative standard deviations were not higher than 2%. Mean recovery was between 98.7 and 101.3%. Calibration curve was linear in the concentration range of 0.5-10 µg/mL. Limits of detection and quantification were 0.19 and 0.60 µg/mL, respectively. Thus, the present report put forward a novel method for the estimation of EAB, an emerging insect repellent, by using RP-HPLC technique.

The Development and Validation of Novel, Simple High-Performance Liquid Chromatographic Method with Refractive Index Detector for Quantification of Memantine Hydrochloride in Dissolution Samples.

The present study was aimed to develop an analytical method for quantification of memantine (MEM) hydrochloride in dissolution samples using high-performance liquid chromatography with refractive index (RI) detector. The chromatographic separation was achieved on C18 (250 × 4.5 mm, 5 μm) column using isocratic mobile phase comprises of buffer (pH 5.2):methanol (40:60 v/v) pumped at a flow rate of 1.0 mL/min. The column effluents were monitored using RI detector. The retention time of MEM was found to be ~6.5 ± 0.3 min. The developed chromatographic method was validated and found to be linear over the concentration range of 5.0-45.0 μg/mL for MEM. Mean recovery of MEM was found to be 99.2 ± 0.5% (w/w). The method was found to be simple, fast, precise and accurate, which can be utilized for the quantification of MEM in dissolution samples.

The simple isocratic HPLC—UV method for the simultaneous determination of reduced and oxidized glutathione in animal tissue

The aim of our work was to optimize and apply simple high-performance liquid chromatography method with ultraviolet detection (HPLC—UV) for simultaneous determination of reduced (GSH) and oxidized (GSSG) glutathione in biological matrix (specifically, the rat liver tissue was used herein), since the ratio between oxidized and reduced glutathione forms (GSSG—GSH) has been recognized as an important biological marker of oxidatively depleted GSH in oxidative stress (OS)-associated diseases and poisonings. An isocratic chromatographic separation of GSH and GSSG (2.8 min and 6.3 min, respectively) was performed with the mobile phase consisted of sodium perchlorate solution (pH adjusted to 2.8) at flow rate of 1 mL min−1, detection set at 215 nm, and column temperature of 40 °C. The method offers short run time, linearity in the range of 0.01—200 μM concentration for both compounds (R2 = 1), low limits of detection and quantification (GSH: 0.18 μM and 0.56 μM, GSSG: 0.52 μM and 1.58 μM, respectively), precision, accuracy (bias < 2%), and high reproducibility. Through suitable sample handling, an overestimation of GSSG was prevented. High recovery (>99%) was achieved. The method was successfully applied for the analysis of GSH and GSSG in liver homogenates of Wistar rats intraperitoneally exposed to cadmium (Cd) (1 mg kg−1 CdCl2/21 days). Regardless of other Cd-mediated hepatotoxicity mechanisms, herein, we have exclusively interpreted/emphasized oxidative GSH depletion. The presented method is acceptable for a routine analysis of GSH and GSSG in biological matrix, while the calculated ratio GSSG—GSH is considered as a valuable OS marker.

Simple Quantification of Pentosidine in Human Urine and Plasma by High-Performance Liquid Chromatography.

Pentosidine is an advanced glycation end-product (AGE) and fluorescent cross-link compound. A simple high-performance liquid chromatographic (HPLC) method was developed for the detection and quantification of pentosidine in human urine and plasma. The mobile phase used a gradient system to improve separation of pentosidine from endogenous peaks, and chromatograms were monitored by fluorescent detector set at excitation and emission wavelengths of 328 and 378 nm, respectively. The retention time for pentosidine was 24.3 min and the lower limits of quantification (LLOQ) in human urine and plasma were 1 nM. The intraday assay precisions (coefficients of variation) were generally low and found to be in the range of 5.19–7.49% and 4.96–8.78% for human urine and plasma, respectively. The corresponding values of the interday assay precisions were 9.45% and 4.27%. Accuracies (relative errors) ranged from 87.9% to 115%. Pentosidine was stable in a range of pH solutions, human urine, and plasma. In summary, this HPLC method can be applied in future preclinical and clinical evaluation of pentosidine in the diabetic patients.

A Validated Stability-Indicating HPLC Method for Simultaneous Determination of Amoxicillin and Enrofloxacin Combination in an Injectable Suspension.

The combination of amoxicillin and enrofloxacin is a well-known mixture of veterinary drugs; it is used for the treatment of Gram-positive and Gram-negative bacteria. In the scientific literature, there is no high-performance liquid chromatography (HPLC)-UV method for the simultaneous determination of this combination. The objective of this work is to develop and validate an HPLC method for the determination of this combination. In this regard, a new, simple and efficient reversed-phase HPLC method for simultaneous qualitative and quantitative determination of amoxicillin and enrofloxacin, in an injectable preparation with a mixture of inactive excipients, has been developed and validated. The HPLC separation method was performed using a reversed-phase (RP)-C18e (250 mm × 4.0 mm, 5 μm) column at room temperature, with a gradient mobile phase of acetonitrile and phosphate buffer containing methanol at pH 5.0, a flow rate of 0.8 mL/min and ultraviolet detection at 267 nm. This method was validated in accordance with the Food and Drug Administration (FDA) and the International Conference on Harmonisation (ICH) guidelines and showed excellent linearity, accuracy, precision, specificity, robustness, ruggedness, and system suitability results within the acceptance criteria. A stability-indicating study was also carried out and indicated that this method can also be used for purity and degradation evaluation of these formulations.

Chromatographic Characterization and Method Development for Determination of Levetiracetam in Saliva: Application to Correlation with Plasma Levels.

Levetiracetam (LVT) is a widely used antiepileptic drug (AED). A less invasive sampling method for therapeutic drug monitoring (TDM) would be very useful particularly for children. Saliva has been shown as an adequate sample for TDM of some AEDs. Due to the high hydrophilicity of LVT its separation on common stationary phases is quite a challenge so that previous methods for determination of LVT in saliva employed either gradient high performance liquid chromatographic (HPLC) system or mass spectrometer as a detector. In this study the retention behavior of LVT on some common stationary phases was examined, with C8 being the most retentive. A simple isocratic HPLC method that is based on simple protein precipitation was developed and validated for the determination of LVT in saliva. The method was applied to a sample group of epileptic children for the purpose of assessing potential correlation with plasma LVT levels and to investigate patient's compliance. The results confirmed a reasonable correlation between plasma and salivary levels of LVT (R = 0.9) which supports the use of saliva for TDM of LVT. The study also revealed a significant percentage of epileptic patients having LVT levels below the estimated therapeutic range.

Development and Validation of A HPLC-UV Method for Dissolution Testing of Ciclosporin: Its Application to The Measurement of Brand and Generic Versions from Different Countries

Ciclosporin is used as an immunosuppressant in post-organ transplantation. Recently, many questions have been raised about using generic substitutes, especially with narrow therapeutic index drugs (NTIDs). In this study, a simple high-performance liquid chromatography (HPLC) method was developed, validated and applied to detection ciclosporin in dissolution testing. Seven ciclosporin products (gelatin capsules) were included in this study, obtained from Columbia (C), Egypt (E), India (I), Jordan (J), Pakistan (P), Saudi Arabia (S), and Turkey (T). The dissolution test was done for all capsules. The Conditions were as follows: 500 ml deionized water as the medium in apparatus 2 (Pharmatest, Germany), temperature 37.5 ± 0.5°C; 50 rev/min, sampling times were 5, 10, 15, 30, 60 and 90 min with 5 ml for each sample. HPLC separation was done by a C18 column, 5 μm, (4.6 × 250 mm, ACE 5) held at 50 ± 0.3°C. Analytes were isocratically eluted at 0.7 ml/min with acetonitrile and water (70+30%) and 0.03% trifluoroacetic acid, over the 25-min run time. The intra-day and inter-day imprecision for ciclosporin across the standard range was <5% and <4%, respectively. The accuracy of the assay was within ± 13% of the true value at standard curve concentrations range from 0.1 to 2 mg/ml of ciclosporin. The lower and the upper limit of detection were 0.001 mg/ ml and 2 mg/ml of ciclosporin, respectively. All brands (S, T, P, J, E) and one generic (C) showed more than 80% of ciclosporin after 90 min (90.3, 100, 90.4, 82.7, 81.4 and 90.6%) respectively. One generic (I), showed less than the minimum percentage of labeled amount, 69.1%. Relative to the brand (T), statistical analysis showed significant differences (P<0.0001) of the mean percentage content between brand and generic. The 95% confidence interval range for the brands (E, J, P, and S) was (72.2-91.8), (73.4-93.3), (80.2-101.9), and (80.1-101.8), respectively, and (80.3-102.1), and (61.3-77.9) for the generic (C) and (I) respectively. Based on these results, we conclude that some of the ciclosporin preparations do not contain the exact mass labeled and the majority contained as yet unidentified impurities.

Development and validation of a simple and robust HPLC method with UV detection for quantification of the hepatitis C virus inhibitor daclatasvir in human plasma

Daclatasvir is an inhibitor of hepatitis C virus NS5A protein that is used for the therapy of chronic hepatitis. So far, published methods for analysis of daclatasvir in plasma are exclusively based on mass spectrometry, which is not always available in standard clinical laboratories. Thus, we wished to develop and validate a simple, but still reliable and sensitive high-performance liquid chromatography (HPLC) assay with UV detection for the quantification of daclatasvir, feasible for a wide-spread clinical routine use. The method consisted of solid-phase extraction of daclatasvir using Waters Oasis HLB 1cc cartridges, reversed-phase liquid chromatography with a Waters XTerra RP18 (150mm×4.6mm, 3.5μm) column and a mobile phase of ammonium acetate buffer (pH 5.0, 10mM) and acetonitrile (56:44, v/v), and UV detection at 318nm. This assay proved to be sensitive (lower limit of quantification of 0.05μg/mL), linear (correlation coefficients ≥0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.9%), and accurate (deviations ranged from −2.2 to 8.0% and from −6.5 to 9.2% for intra-day and inter-day assays, respectively). The method was applied to therapeutic monitoring of patients undergoing daclatasvir therapy for hepatitis C and showed to be reliable and robust. Thus, this method provides a simple, sensitive, precise, and reproducible assay for dosing daclatasvir that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, the stability of daclatasvir in plasma was evaluated under various conditions, including after the heating procedure required for inactivation of infectious viruses and in different light exposure conditions. These studies evidenced photo-instability of the compound under sunlight exposure over time. Thus, blood sampling and the whole handling procedure have to be performed quickly and with minimal light exposure.

An easy-to-use liquid chromatography assay for the analysis of lamotrigine in rat plasma and brain samples using microextraction by packed sorbent: Application to a pharmacokinetic study.

A simple and rapid high-performance liquid chromatography method with diode-array detection (HPLC-DAD) using microextraction by packed sorbent (MEPS) during the sample preparation step was developed and validated to quantify lamotrigine (LTG) in rat plasma and brain samples. MEPS variables such as pH, number of draw-eject cycles, and washing and desorption conditions were optimized. The chromatographic resolution of LTG and chloramphenicol, used as internal standard (IS), was accomplished in less than 5min on a C18 column, at 35°C, using an isocratic elution with acetonitrile (13%), methanol (13%) and water-triethylamine (99.7:0.3, v/v; pH 6.0) pumped at a flow rate of 1mL/min. Detection was performed at 215nm. Calibration curves were linear over the range of 0.1-20μg/mL (r2≥0.9947) for LTG in both rat plasma and brain homogenate samples. The intra and interday imprecision did not exceed 8.6% and the intra and interday inaccuracy ranged from -8.1 to 13.5%. LTG was extracted from rat plasma and brain homogenate samples with an average absolute recovery ranging from 68.0 to 86.7%, and its stability was demonstrated in the assayed conditions. No interferences were observed at the retention times of the analyte (LTG) and IS. To the best of our knowledge, this is the first bioanalytical assay that uses MEPS procedure for the determination of LTG not only in rat plasma but also in tissue (brain) samples. This novel method was successfully applied to a preliminary pharmacokinetic study in rats and it seems to be a cost-effective tool to support non-clinical pharmacokinetic-based studies involving LTG treatment.

Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride.

Besifloxacin is a unique chiral broad-spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R-form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S-isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high-performance liquid chromatography (HPLC) method for determination of R-besifloxacin and S-besifloxacin (BES impurity A) was developed and validated for in-process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD-H (250 x 4.6mm, 5μm) column using n-heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290nm. The resolution between S-isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal-to-noise ratio of 3 and 10 the LOD of besifloxacin was 0.30μg/mL, while the LOQ was 0.90μg/mL. The calibration curves were linear in the range of 0.9-7.5μg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628-632, 2016. © 2016 Wiley Periodicals, Inc.

Simple high‐performance liquid chromatography method for formaldehyde determination in human tissue through derivatization with 2,4‐dinitrophenylhydrazine

A simple high-performance liquid chromatography method has been developed for the determination of formaldehyde in human tissue. FA Formaldehyde was derivatized with 2,4-dinitrophenylhydrazine. It was extracted from human tissue with ethyl acetate by liquid-liquid extraction and analyzed by high-performance liquid chromatography. The calibration curve was linear in the concentration range of 5.0-200μg/mL. Intra- and interday precision values for formaldehyde in tissue were <6.9%, and accuracy (relative error) was better than 6.5%. The extraction recoveries of formaldehyde from human tissue were between 88 and 98%. The limits of detection and quantification of formaldehyde were 1.5 and 5.0μg/mL, respectively. Also, this assay was applied to liver samples taken from a biopsy material.

Determination of Urine Albumin by New Simple High-Performance Liquid Chromatography Method.

A simple high-performance liquid chromatography (HPLC) method was developed for the determination of albumin in patients' urine samples without coeluting proteins and was compared with the immunoturbidimetric determination of albumin. Urine albumin is important biomarker in diabetic patients, but part of it is immuno-nonreactive. Albumin was determined by high-performance liquid chromatography (HPLC), UV detection at 280 nm, Zorbax 300SB-C3 column. Immunoturbidimetric analysis was performed using commercial kit on automatic biochemistry analyzer COBAS INTEGRA® 400, Roche Diagnostics GmbH, Manheim, Germany. The HLPC method was fully validated. No significant interference with other proteins (transferrin, α-1-acid glycoprotein, α-1-antichymotrypsin, antitrypsin, hemopexin) was found. The results from 301 urine samples were compared with immunochemical determination. We found a statistically significant difference between these methods (P = 0.0001, Mann-Whitney test). New simple HPLC method was developed for the determination of urine albumin without coeluting proteins. Our data indicate that the HPLC method is highly specific and more sensitive than immunoturbidimetry.

Development and Validation of Reverse-phase High-performance Liquid Chromatography Method for Estimation of Citicoline Sodium in Bulk and Dosage Form.

A simple, accurate, precise and sensitive reverse-phase high-performance liquid chromatography method for the determination of citicoline sodium has been developed and validated. Drug was resolved on a C18 column (Phenomenex Luna, 250×4.6 mm, 10μ), utilizing mobile phase of potassium dihydrogen phosphate buffer and acetonitrile in a ratio of 30:70. Mobile phase was delivered at the flow rate of 1.0 ml/min and detection was carried out at 272 nm. Separation was completed within 2.22 min. Calibration curve was linear with good correlation coefficient (R(2)=0.999) over a concentration range 10-60 μg/ml. Recovery was between 98.84 and 101.76%. Method was found to be reproducible with relative standard deviation for intra and interday precision of <2.0% over the said concentration range. The method was successfully applied to the determination of the citicoline sodium, it can be very useful and an alternate to performing the stability studies.

Development and validation of HPLC method for vicenin-1 isolated from fenugreek seeds in rat plasma: application to pharmacokinetic, tissue distribution and excretion studies

Context: Vicenin-1, a flavonol glycoside, has potent platelet aggregation inhibition, antioxidant, radioprotectants and anti-inflammatory activities.Objective: To establish a rapid, simple, precise and sensitive high-performance liquid chromatography (HPLC) method for determination of vicenin-1 in rat plasma, and to investigate the pharmacokinetics, tissue distribution and excretion after a single 60 mg/kg oral dose in rats.Materials and methods: Vicenin-1 was extracted by solid–liquid extraction through Tulsicon® ADS-400 (0.40–1.2 mm). Chromatographic separation was achieved by HPLC with a C18 column with a mobile phase composed of water and acetonitrile (75:25 v/v) and a flow rate of 1 mL/min along with UV detection at 210 nm.Results: Good linearity of calibration curve was found between 10.5 and 100.5 μg/mL (R2 = 0.995) for plasma and tissue, whereas 2.5–500 μg/mL (R2 = 0.999) for the urine and stool samples. The extraction recoveries were 98.51–99.58% for vicenin-1 in plasma, whereas intra-day and inter-day precision were validated by relative standard deviation (%RSD), that came in the ranges of 1.16–1.79% and 1.28–1.73%, respectively. The pharmacokinetics results showed Cmax (7.039 μg/mL) and Tmax (2 h) after oral administration of vicenin-1. Tissue distribution study showed that the highest concentration of vicenin-1 was achieved in the liver followed by the lung. Approximately 24.2% of its administered dose was excreted via urinary excretion route.Conclusion: The first-pass metabolism, poor solubility and presence of reducing sugar moiety in vicenin-1 may decrease its bioavailability. The developed method is sensitive, specific and was successfully applied to the pharmacokinetics, tissue distribution and excretion studies of vicenin-1 in rats.

Development of an HPLC Method with an ODS Column to Determine Low Levels of Aspartame Diastereomers in Aspartame.

α-L-Aspartyl-D-phenylalanine methyl ester (L, D-APM) and α-D-aspartyl-L-phenylalanine methyl ester (D, L-APM) are diastereomers of aspartame (N-L-α-Aspartyl-L-phenylalanine-1-methyl ester, L, L-APM). The Joint FAO/WHO Expert Committee on Food Additives has set 0.04 wt% as the maximum permitted level of the sum of L, D-APM and D, L-APM in commercially available L, L-APM. In this study, we developed and validated a simple high-performance liquid chromatography (HPLC) method using an ODS column to determine L, D-APM and D, L-APM in L, L-APM. The limits of detection and quantification, respectively, of L, D-APM and D, L-APM were found to be 0.0012 wt% and 0.004 wt%. This method gave excellent accuracy, repeatability, and reproducibility in a recovery test performed on five different days. Moreover, the method was successfully applied to the determination of these diastereomers in commercial L, L-APM samples. Thus, the developed method is a simple, useful, and practical tool for determining L, D-APM and D, L-APM levels in L, L-APM.