RP-LC-UV Determination of Proanthocyanidins in Guazuma ulmifolia

A simple, reproducible, and efficient liquid chromatographic method was developed with UV detection. Water (0.05% TFA):acetonitrile (0.05% TFA) was used as the mobile phase in a gradient system for the determination of procyanidin B2 (PB2) and epicatechin (EC) in the bark of Guazuma ulmifolia Lam. The analysis was performed using a Phenomenex Gemini RP C₁₈ column (5 μm) as stationary phase, at 30 °C, with a flow rate of 0.8 mL min⁻¹, at a wavelength of 210 nm for detection and determination. The main validation parameters of the method were also determined. Calibration curves were found to be linear, with ranges of 20.00–150.00 (PB2) and 10.00–110.00 μg mL⁻¹ (EC). The correlation coefficients of linear regression analysis were between 0.9981 and 0.9988, and the detection limits were between 2.89 and 2.54 μg mL⁻¹. The contents of PB2 and EC were successfully determined, with satisfactory reproducibility and recovery. Recoveries of the PB2 and EC were 103.00 and 104.01%, respectively. The method was successfully applied to the determination of procyanidins in the bark of G. ulmifolia.     

Determination of Three Nonsteroidal Anti-Inflammatory Drugs in Human Plasma by LC Coupled with Chemiluminescence Detection

A simple and sensitive method was developed for the determination of three nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, naproxen and fenbufen in human plasma. The method involved in column liquid chromatographic separation and chemilumenescence (CL) detection based on the CL reaction of NSAIDs, potassium permanganate (KMnO₄) and sodium sulfite (Na₂SO₃) in sulfuric acid (H₂SO₄) medium. The chromatographic separation was carried out using a reversed-phase C₁₈ column, which allowed the selective determination of the three medicines in the complicated samples. The special features of the CL detector provided lower LOD for determination than that of existing chromatographic alternatives. The results indicated that the linear ranges were 0.01–10.0 μg mL⁻¹ for ibuprofen, 0.001–1.0 μg mL⁻¹ for naproxen, and 0.01–10.0 μg mL⁻¹ for fenbufen. The limits of detection were 0.5 ng mL⁻¹ for ibuprofen, 0.05 ng mL⁻¹ for naproxen and 0.5 ng mL⁻¹ for fenbufen (S/N = 3). All average recoveries were in the range of 90.0–102.3%. Finally, the method had been satisfactorily applied for the determination of ibuprofen, naproxen and fenbufen in human plasma samples.

     

Determination of Sulfonamides in Pharmaceuticals and Rabbit Plasma by Microchip Electrophoresis with LED-IF Detection

In this paper, we describe a compact and low-cost light-emitting diode-induced fluorescence (LED-IF) detection coupled to microchip electrophoresis for the determination of sulfonamides in pharmaceutical formulations and rabbit plasma. Three fluorescein isothiocyanate-labeled sulfonamides in rabbit plasma were separated in the running buffer of 40 mM phosphate buffer (pH 7.0) at the separation voltage of 2.0 kV, and detected by LED-IF detector in which the high-power blue LED was driven at the constant current of 150 mA and the emitted fluorescence over 510 nm was collected by a planar photodiode. The linear concentration ranged from 2.0 to 125.0 μg mL⁻¹, both for sulfadiazine and sulfamethazine with the correlation coefficients (r ²) of 0.995 and 0.997, respectively, and from 2.0 to 100.0 μg mL⁻¹ with the correlation coefficients (r ²) of 0.997 for sulfaguanidine. The limits of detection for the three sulfonamides were 0.36–0.50 μg mL⁻¹ (S/N = 3). Intra-day and inter-day precision of migration time and peak area for the determination of sulfonamides were <4.5 %. This method has been successfully applied to the analysis of sulfonamides in pharmaceuticals, and could be used to study the pharmacokinetics of sulfonamides in rabbit.

     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL⁻¹ of ENL, 0.260–399 μg mL⁻¹ of HCZ for HPLC system and 0.270–399 μg mL⁻¹ of ENL and 0.065–249 μg mL⁻¹ of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL⁻¹ and 31.477 ng mL⁻¹ for HCZ, 2.804 ng mL⁻¹ for ENL and 2.943 ng mL⁻¹ for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.

     

Development and Validation of a Stability-Indicating HPLC Method for Determination of Ciprofloxacin Hydrochloride and its Related Compounds in Film-Coated Tablets

The objective of the current study was the development and subsequent validation of a simple, sensitive, precise and stability-indicating reversed-phase HPLC method for the determination of ciprofloxacin HCl in pharmaceutical dosage forms in the presence of its potential impurities. The chromatographic separation of ciprofloxacin HCl and its related compounds was achieved on an Inertsil ODS3 column using UV detection. The optimized mobile phase consisted of phosphoric acid solution: acetonitril. The proposed method provided linear responses within the concentration range 250–750 μg mL⁻¹ for ciprofloxacin HCl and 0.5–1.5 μg mL⁻¹ for its related compounds. LOD and LOQ values for the active substance were 5.159 and 15.632 μg mL⁻¹, respectively. Correlation coefficients (r) of the regression equations for the impurities were greater than 0.99 in all cases. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed.

     

Simultaneous Determination of Antibiotics in Anti-Acne Cosmetics by Rapid LC with DAD

A suitable method that allows, for the first time, the simultaneous determination of nine antibiotics which may help the therapy of acne vulgaris by rapid liquid chromatography with diode array detection in 7 min is presented in this work. An SB RP18 (50 × 4.6 mm; 1.8 μm particle size) column was used with the mobile phase consisting of a mixture of 0.1 mol L⁻¹ potassium dihydrogen phosphate (pH 2.5) and acetonitrile at the gradient elution program. The correlation coefficients were all above 0.9999 in the linear range between 4–100 μg mL⁻¹, the average spiked recoveries (n = 6) were 92.2–103.2% with RSD ranging from 0.04 to 4.5% depending on the target analytes. The method detection limits were in the range of 0.02–0.2 μg mL⁻¹ in anti-acne cosmetics. The analysis of real cosmetic preparations demonstrated the fitness for the whole analytical procedure. The proposed method appeared therefore as a sound alternative for official testing method, which could overcome the general problems of time consuming, lack of the specificity and precision difficulty.

     

Determination of Repertaxin Enantiomeric Purity by HPLC on Chiral Stationary Phase

A precise and sensitive LC method for the determination of repertaxin enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 2.0 was accomplished within 20 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane:2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min⁻¹. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition, temperature and flow rate on enantiomeric selectivity and on resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.002 and 1.0 mg mL⁻¹ (n = 3), and relative standard deviation (RSD) of the inter-batch assay and intra-batch assay was less than 1.27 and 1.16 %. LOD and LOQ for repertaxin were 0.65 and 2.19 μg mL⁻¹; those for its enantiomer were 0.70 and 2.34 μg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of repertaxin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

Development of a Stability-Indicating LC Assay Method for Determination of Chloroquine

A simple and rapid development of a stability-indicating LC method for determination of chloroquine diphosphate in the presence of its hydrolysis, oxidative and photolysis degradation products is described. Stress testing showed that chloroquine diphosphate was degraded under basic conditions and by photolytic treatment but was stable under the other stress conditions investigated. Separation of the drug from its degradation products was achieved with a Nova Pack C18 column, 0.01 M PIC B7 and acetonitrile (40:60 v/v) pH 3.6, as mobile phase. Response was linear over the range 0.08–5.70 μg mL⁻¹ (r = 0.996), with limits of detection and quantification (LOD and LOQ) of 0.17 and 0.35 μg mL⁻¹, respectively.

     

Determination of Tectorigenin in Rat Plasma: Application to a Pharmacokinetic Study After Oral Administration of Tectorigenin or Its Prodrug Tectoridin

A simple, sensitive, and validated liquid chromatographic method has been developed for the determination of tectorigenin in rat plasma and application to a pharmacokinetic study after oral administration of tectorigenin or its prodrug tectoridin. The analysis was performed on a Kromasil C₁₈ analytical column using gradient elution with acetonitrile 0.1% phosphonic acid water at 0.8 mL min⁻¹. The detection wavelength for UV detection was set at 264 nm. The established method was fully validated with parameters as follows: the intra- and inter-day assay precisions (CV) of three analytes were in the range of 4.2–13.3% and accuracies were between 98.0 and 107.5%; the calibration curve was linear with r ² > 0.99 over a concentration range of 0.02–2 μg mL⁻¹; the lower limit of quantification was 0.02 μg mL⁻¹; tectorigenin showed stable in rat plasma after 12 h incubation at room temperature, 15 days storage at −80 °C and three freeze/thaw cycles, as well as in reconstitute buffer for 24 h at 25 °C; and the mean recoveries of tectorigenin were 92.3 ± 3.2, 95.5 ± 2.9 and 94.5 ± 3.0% with quality control levels of 0.02, 0.2 and 2 μg mL⁻¹, respectively. In conclusion, this method is simple, economic, and sensitive enough for in vivo pharmacokinetic studies of tectorigenin.

     

Quantitation of Eugenol,Cinnamaldehyde and Isoeugenol from Cinnamomum tamala Nees and Eberm. Leaf Powder and Cinnamomum zeylanicum Breyn Stem Bark Powder by LC

An LC method has been developed for the simultaneous quantitative determination of eugenol, cinnamaldehyde and isoeugenol from dried leaf powder of Cinnamomum tamala Nees & Eberm and stem bark powder of Cinnamomum zeylanicum Breyn. Linear responses for eugenol, cinnamaldehyde and isoeugenol were obtained over the concentration ranges of 0.20–2.50, 5.00–100.00 and 0.10–1.00 μg mL⁻¹, respectively.

     

HPLC-ELSD Determination of Kryptofix 2.2.2 in the Preparations of 2-Deoxy-2-[18F]fluoro-d-glucose and other Radiopharmaceuticals

A new and simple high-performance liquid chromatography with evaporative light scattering detector method for the determination of Kryptofix 2.2.2 (K-222) in the radiopharmaceuticals of 2-deoxy-2-[¹⁸F]fluoro-d-glucose ([¹⁸F]FDG) and 3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT) was developed. A C₁₈ column was used and the mobile phase was 10 % (v/v) methanol and 90 % (v/v) water (0.1 % trifluoroacetic acid, v/v) at a flow rate of 0.2 mL min⁻¹. The drift tube temperature was 40 °C. The pressure of nebulizing gas (N₂) was 3.0 bar. The gain was 10. Good separation of K-222 from main related substances could be achieved. Excellent linearity (r ² = 0.9995) was obtained over the range of 5–100 μg mL⁻¹. The precision ranged from 0.68 to 5.16 % (RSD) and the accuracy ranged from −3.05 to 2.62 % (RE). The limit of detection was 2 μg mL⁻¹. This method offers simple, rapid and quantitative detection of K-222, thus making it acceptable for routine determination.

     

Development and Validation of a Stability-Indicating LC Method for the Determination of Rupatadine in Pharmaceutical Formulations

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of rupatadine in pharmaceutical dosage forms. The LC method was carried out on a Gemini C₁₈ column (150 mm × 4.6 mm I.D.), maintained at 30 °C. The mobile phase consisted of ammonium acetate buffer (pH 3.0; 0.01 M) with 0.05% of 1-heptanesulfonic acid–acetonitrile (71.5:28.5, v/v), run at a flow rate of 1.0 mL min⁻¹ and using photodiode array (PDA) detection at 242 nm. The chromatographic separation was obtained with retention time of 5.15 min, and was linear in the range of 0.5–400 μg mL⁻¹ (r ² = 0.9999). The specificity and stability-indicating capability of the method was proven through the degradation studies and showing also, that there was no interference of the excipients. The accuracy was 100.39% with bias lower than 0.58%. The limits of detection and quantitation were 0.01 and 0.5 μg mL⁻¹, respectively. Moreover, method validation demonstrated acceptable results for precision, sensitivity and robustness. The proposed method was applied for the analysis of pharmaceutical dosage forms assuring the therapeutic efficacy.

     

Development of a Validated Stability-Indicating LC Assay and Stress Degradation Studies of Linezolid in Tablets

This paper describes the validation of an isocratic LC method for the assay of linezolid in tablets. Validation parameters such as linearity, precision, accuracy, specificity, limit of detection, limit of quantitation and robustness were determined. LC was carried out by reversed phase technique on an RP-18 column with a mobile phase composed of 1% acetic acid:methanol:acetonitrile (50:25:25, v/v/v). Linezolid and your combination drug product were exposed to acid, base, oxidation, dry heat and photolytic stress conditions. A linear response (r > 0.9999) was observed in the range of 8.0–20.0 μg mL⁻¹. The retention time of linezolid was 4.6 min. The method showed good recoveries and intra- and inter-day relative standard deviations were less than 1.0%. The LOD and LOQ were 0.21 and 0.63 μg mL⁻¹, respectively. The developed LC method for determination of related substances and assay determination of linezolid can be used to evaluate the quality of regular production samples. It can also be used to test the stability samples of linezolid.

     

Determination of Hg(II) in Environmental Water Samples Using DLLME Method Prior to GC-FID

Mercury exists in two forms in environment, inorganic salts and organic compounds. Determination of mercury is very important, due to its health effects. In the present research, diphenylation of mercury using phenylboronic acid as a derivatization reagent was used for the determination of Hg(II) in real water samples. A simple, rapid and cheap method named dispersive liquid–liquid microextraction was used for the extraction of analyte under the following conditions: extraction solvent 16 μL of carbon tetrachloride, disperser solvent 1 mL of ethanol and sample volume 5 mL. Under the optimal conditions, the enrichment factor for diphenylmercury was 931 and the limit of detection calculated on the basis of five replicates was 0.004 μg mL⁻¹. The repeatability of the method expresses as relative standard deviation was 5.1 (n = 6). The linear range was between 0.01 and 10 μg mL⁻¹. The performance of the proposed technique was evaluated for the determination of mercury in different environmental water samples.

     

LC Method for Quantification of ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Rabbit Plasma: Validation and Application to a Pharmacokinetic Study

ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic acid (5F), a diterpenoid isolated from the Chinese herb Pteris semipinnata L, has been suggested to show antitumor properties. A simple and sensitive LC method was developed for the determination of 5F in rabbit plasma. The method involved liquid–liquid extraction using ethyl acetate under acidic conditions using naproxen as an internal standard. Separations were performed on a reversed-phase column with a mixture of 1% (v/v) glacial acetic acid and methanol (45:55, v/v) as mobile phase and UV detection was utilized at 242 nm. The calibration plot was linear in the range 0.20–10.0 μg mL⁻¹ (correlation coefficients r ² > 0.998). The detection limit was 0.20 μg mL⁻¹, mean extraction recovery was above 82%, intra-day precision of the method was less than 6.4%, and inter-day precision was better than 8.7%, respectively. The validated assay was found to be suitable for the pharmacokinetic study of 5F in rabbits.

     

A Rapid Determination of Taurine in Human Plasma by LC

Taurine is an amino acid which is not incorporated into proteins but found in the cytosol of many mammalian cells, in high concentrations (2–30 mM). Increase in plasma taurine concentration has already been reported after surgical trauma, X-radiation, muscle necrosis, carbon tetrachloride-induced liver damage, and paracetamol overdose. Plasma taurine concentration was measured using LC with fluorescence detection following derivatization by o-phtalaldehyde plus 3-mercapto-propionic acid and α-aminobutyric acid as internal standard. Under these conditions the retention time of taurine was 10 min. This method was sensitive enough, to quantify 150 pg mL⁻¹ and detect 50 pg mL⁻¹ of taurine ranging normally between 65 and 179 mmol L⁻¹ (8–22 μg mL⁻¹). The validated method allowed simple determination of human plasma taurine in pharmacokinetic and biomarker studies.

     

Simultaneous HPLC–DAD Determination of Retinol and Eight Vitamin E Isomers in Human Serum

An efficient, high-performance liquid-chromatographic method with diode-array detection (HPLC–DAD) has been established for simultaneous determination of retinol, α, (β + γ), and δ-tocopherols, and α, β, γ, and δ-tocotrienols in human serum. After deproteinization, the target vitamins in serum were extracted with n-hexane and the extract was evaporated under weak nitrogen flow. The residue was redissolved in methanol and the resulting solution was used for HPLC analysis. Retinol acetate and α-tocopherol acetate were used as internal standards. The internal standard calibration curves were linear over the range of 0.010–50.0 µg mL⁻¹, with correlation coefficients >0.999. Mean recoveries of the method were 86.3–110 %, with intra-day and inter-day relative standard deviations less than 12.2 and 14.9 %, respectively. The detection limits of the method ranged from 0.001 to 0. 002 µg mL⁻¹, and the quantification limits ranged from 0.002 to 0.008 µg mL⁻¹. The method was successfully applied to analysis of the target vitamins in 50 human serum samples; all the analytes were detected at concentrations ranging from <0.002–23.0 µg mL⁻¹.

     

Application of Multicriteria Methodology in the Development of Improved RP-LC-DAD for Determination of Rizatriptan and Its Degradation Products

A simple, rapid, and stability-indicating reversed-phase high-performance liquid chromatographic (LC) method for analysis for dutasteride has been successfully developed. Chromatography was performed on a 150 mm × 4.6 mm C₁₈ column with acetonitrile–water 60:40 (v/v) as isocratic mobile phase at 1.0 mL min⁻¹. Ultraviolet detection of dutasteride was at 210 nm. Its retention time was approximately 10 min and its peak was symmetrical. Response was a linear function of concentration over the range 0.2–1 μg mL⁻¹ (R ² = 0.997) and the limits of detection and quantitation were was 0.05 and 0.10 μg mL⁻¹, respectively. The method was validated for linearity, precision, repeatability, sensitivity, and selectivity. Selectivity was validated by subjecting dutasteride stock solution to photolytic, acidic, basic, oxidative, and thermal degradation. The peaks from the degradation products did not interfere with that from dutasteride. The method was used to quantify dutasteride in pharmaceutical preparations.

     

Pharmacokinetics and Tissue Distribution Study of Tryptanthrin in Rats by RP-HPLC with DAD Detector

A simple RP-LC-UV method was established for the determination of tryptanthrin in plasma and different tissues of rats. The separation was achieved by HPLC on a C₁₈ column with a mobile phases composed of acetonitrile–water (47:53, v/v), UV detection was used at 251 nm. Good linearity was found between 0.0183–1.1712 μg mL⁻¹ (r ² = 0.999) for plasma and 0.0937–1.7568 μg mL⁻¹ for the tissue samples, respectively (r ² ≥ 0.9932). The intra- and inter-day precisions expressed as the relative standard deviation for the method were 0.92–6.01 and 1.06–9.11 %, respectively. The relative recoveries of tryptanthrin ranged from 95.26 to 97.89 % for plasma and 82.55 to 114.99 % for tissue homogenates (except heart). The developed method was successfully applied to the pharmacokinetics and tissue distribution research after orally administration of a 56-mg kg⁻¹ dose of tryptanthrin to healthy SD rats. The main pharmacokinetics distribution results showed that liver, lung, small intestine, and large intestine were the major distribution tissues of tryptanthrin in rats, and that tryptanthrin had difficulty in crossing the blood–brain barrier.