Optimization of Microwave-Assisted Extraction for the Determination of Glycyrrhizin in Menthazin Herbal Drug by Experimental Design Methodology

In the present study, a microwave-assisted extraction (MAE) method has been investigated for the extraction of glycyrrhizin from Menthazin herbal drug. The extracted samples have been analyzed by a developed reversed-phase liquid chromatography with ultraviolet detection. The separation was performed by a Eurospher-100 C₈ reversed-phase column (250 × 4.6 mm i.d., 5 μm) and the mobile phase consisted of methanol:acetonitrile:water:glacial acetic acid (30:30:40:1 v/v/v/v) with a flow rate of 0.8 mL min^?1. The extraction procedure has been screened by a two level full factorial design for determination of statistically significant parameters. Thereafter, the identified parameters, extraction temperature, time and solvent volume were optimized by a Box–Behnken design. The proposed mathematical model was based on analysis of variance results and correctly explained the behavior of the response in the experimental domain. R ² value adjusted for numbers of degrees of freedom was 0.9915 and P-value for lack of fit, 0.8499 at the 95% confidence level, P > 0.05. The optimal condition identified were extraction temperature, 70 °C, time, 13.8 min and solvent volume 2.0 mL. To evaluate the applicability of the proposed MAE method, results were compared with those obtained with the liquid extraction method. Extraction efficiency and precision were higher when MAE has been used. The proposed method allows extracting the glycyrrhizin in a small quantity of solvent and faster than the liquid extraction method.     

Determination of aminoglutethimide enantiomers in pharmaceutical formulations by capillary electrophoresis using methylated-β-cyclodextrin as a chiral selector and computational calculation for their respective inclusion complexes

A capillary electrophoretic method for the separation of the aminoglutethimide (AGT) enantiomers using methylated-β-cyclodextrin (M-β-CD) as chiral selector is described. Several parameters affecting the separation were studied, including the type and concentration of chiral selector, buffer pH, voltage and temperature. Good chiral separation of the racemic mixture was achieved in less than 9 min with resolution factor Rs = 2.1, using a fused-silica capillary and a background electrolyte (BGE) of tris-phosphate buffer solution (50 mmol L⁻¹, pH 3.0) containing 30 mg mL⁻¹ of M-β-CD. The separation was carried out in normal polarity mode at 25 °C, 16 kV and using hydrostatic injection. Acceptable validation criteria for selectivity, linearity, precision, and accuracy/recovery were included. The proposed method was successfully applied to the assay of AGT enantiomers in pharmaceutical formulations. The computational calculations for the inclusion complexes of the R- and S-AGT-M-β-CD rationalized the reasons for the different migration times between the AGT enantiomers.     

Spectrophotometric procedure for indirect determination of ranitidine in pharmaceutical formulation using fluorescein sodium

A simple and sensitive spectrophotometric method for the determination of ranitidine hydrochloride (R·HCl) in pharmaceutical formulation is proposed. The procedure is based on the oxidation of R·HCl by bromine, generated in situ by the action of bromate?Cbromide mixture in acid medium, followed by estimation of surplus oxidant by its reaction with fluorescein sodium salt (FL). The decrease in concentration of FL is estimated by measuring its absorbance at ?? _max?=?436?nm. All variables affecting the reaction conditions, such as concentration of NaBrO₃, HCl, NaBr and FL, and reaction time were carefully studied and optimized. The analytical curve was linear in the R·HCl concentration range from 0.3 to 8???g?mL^?1 with a detection limit of 0.13???g?mL^?1. The reliability of the proposed spectrophotometric method was established by parallel determination of pure and dosage forms containing R·HCl, by the reference method and by recovery studies.     

Determination of free and total warfarin concentrations in plasma using UPLC MS/MS and its application to a patient samples

Warfarin is routinely monitored by assessing its pharmacologic effects on the international normalized ratio. However, having a patient with INR not responding to increasing warfarin dose mandates a direct measurement of warfarin concentrations (total and free) for better patient clinical management of warfarin therapy. Therefore, a new fully validated specific, precise and accurate ultra-performance liquid chromatography tandem mass spectrometry was developed for the determination of free and total warfarin in human plasma. Free warfarin was measured in plasma filtrate, prepared by ultrafiltration, and sample pretreatment involved protein precipitation with acetonitrile. Linear response (r(2) ≥0.99) was observed over the studied range of free and total warfarin, with the lower limit of detection of 0.25 ng/mL. The intra- and inter-day precision (relative standard deviation) values were <10% and the accuracy (relative error) was ≤6.6 for free and total warfarin. There was no significant difference (p>0.05) between inter- and intra-day studies for the free and total warfarin, which confirmed the reproducibility of the assay method. The mean extraction efficiency was 88.6-107.2% of free and total warfarin. The assay was sensitive to follow warfarin pharmacokinetics (free and total) in a patient with resistance to warfarin up to 24 h after a daily dose of warfarin.     

Enantiomeric resolution of ibuprofen and flurbiprofen in human plasma by SPE-chiral HPLC methods

Chiral analysis of profens in human plasma is an important area of research due to different pharmaceutical activities of their enantiomers. The solid phase extraction of ibuprofen and flurbiprofen from human plasma was carried out on C18 cartridges by using phosphate buffer (50 mM, pH 6.0) followed by elution with methanol. Chiral-HPLC was performed on AmyCoat RP (150 mm x 46 mm, 3 μm particle size) column by using different combinations of water-acetonitrile-trifluoro acetic acid at 1.5 mLmin-1 flow rate. The detection was achieved at 236 and 254 nm for ibuprofen and flurbiprofen, respectively with 27±1°C as working temperature. The chromatographic parameters i.e. retention (k), separation (α) and resolution (Rs) factors ranged from 4.54-14.42, 1.10-1.30 and 1.01-1.49, respectively. The binding differences of enantiomers of ibuprofen and flurbiprofen were 4.4 and 5.2, respectively. These values suggest that S-(+)- enantiomer of flurbiprofen is more active than ibuprofen due to low enantiomeric difference of the later drug. The developed SPE-Chiral HPLC methods were validated, which are selective, efficient and reproducible.     

Enantiomeric resolution of some imidazole antifungal agents on chiralpak WH chiral stationary phase using HPLC

Summary The enantiomeric resolution of (±)-econazole, (±)-miconazole and (±)-sulconazole was achieved on a Chiralpak WH column. The mobile phase used was hexane-2-propanol-diethylamine (400:99:1,v/v/v). The flow rates of the mobile phase used were 0.50 and 1.00 mL min⁻¹. The values of α of the resolved enantiomers of econazole, miconazole and sulconazole were in the range of 1.68 to 1.23 while the values of Rs varied from 2.42 to 1.10. The resolution of these antifungal agents on Chiralpak WH column is governed by ligand exchange mechanism. Hydrophobic interactions also play an important role for the enantiomeric resoltuion of antifungal agents on the reported CSP.     

Advantages of USP Apparatus IV (flow-through cell apparatus) in dissolution studies

The basic characteristics of the flow-through cell apparatus (USP Apparatus IV) including the assembly and open/closed configuration of the apparatus have been described. The relative advantages of the flow-through cell apparatus over other release setups have been summarized. Finally, potential applications of this setup are presented.     

Clinical reference materials for the validation of the performance of photometric systems used for clinical analyses

 There are a wide variety of spectrophotometric devices nowadays used in health services with various qualities of manufacture methods of measurement and metrological characteristics for performing the necessary measurements. Therefore, to meet the accuracy and repeatability requirements needed in medical diagnosis and treatment, the validation of the performance of such systems by clinical chemistry laboratories is essential. However, the validation of a spectrophotometric system for clinical analyses requires several reference materials, according to the end use of the measurement results. This paper discusses some characteristics required of the clinical reference materials needed and used by Romanian Institute of Metrology for validation work. Types of clinical reference materials developed in the national area for this purpose are also presented. Received: 23 April 1997 · Accepted: 7 July 1997     

Some practical aspects of the validation of photometric systems for clinical analyses

 It is well known that erroneous data reported to a physician may strongly affect medical decision making. For routine clinical chemistry purposes, different instrumentation can be used to compare measurements of unknown samples with standard reference materials. Currently, acceptable limits of accuracy and precision are poorly defined in the field of clinical chemistry laboratories. In this article, problems associated with spectrophotometric measurements, both manual and automated, are discussed. The task of the validation of photometric systems for clinical analyses is currently of considerable interest. Some practical aspects of this validation and the use of reference materials for this activity in the national area are discussed. Received: 7 November 1996 Accepted: 14 January 1997     

Quantitative determination of clenbuterol enantiomers in human plasma by high-performance liquid chromatography using the macrocyclic antibiotic chiral stationary phase teicoplanin

We report a method for the high-performance liquid chromatographic (HPLC) chiral separation of racemic clenbuterol in human plasma. Human plasma was spiked with stock solutions of clenbuterol hydrochloride and practolol as the internal standard. Following a liquid-liquid extraction procedure with 10% (+/-)-2-butanol/isopropyl ether under alkaline conditions, the dried samples were reconstituted in methanol and chromatographed using a macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T(trade mark) (teicoplanin). The mobile phase composition was methanol:acetonitrile (70:30, v/v), containing 0.3% (v/v) acetic acid and 0.2% (v/v) triethylamine. The resulting chromatogram achieved baseline separation for the clenbuterol enantiomers. Calibration curves (peak area ratio vs plasma concentration, n = 10) were constructed for the (-)-R-and (+)-S-clenbuterol enantiomers with a plasma concentration range of 0. 25-10 microM. The correlation coefficient (r) range was 0.99988-0. 99999 (mean = 0.99999). The lowest concentration measured was 0.25 microM. Inter- and intra-assay variation was determined for the lowest, medium and highest plasma concentration (0.25, 2 and 10 microM) by calculating the analytical recoveries with a range of 96-104%. The percentage recoveries for the clenbuterol enantiomers were 88.4-102% over the concentration range used. Detailed methodology is presented.