Normal phase thin layer chromatography for aromatic derivatives of 3-chloro-1,4-naphtochinone

The chromatographic characteristics were investigated for eight aromatic derivatives of 3-chloro-1,4-naphthoquinone under conditions of normal-phase thin-layer chromatography for benzene-based binary mobile phase and such polar solvents as chloroform, acetone, acetonitrile, methanol and propan-2-ol. The slope of linear retention dependencies for the investigated compounds on the concentration of the polar component in the mobile phase satisfactorily correlates with the area occupied by the adsorbed analyte molecule in the stationary phase. The intercept in the Soczewinski equation depends on the polar component of the mobile phase.

DEVELOPMENT AND VALIDATION FOR FREE AGLYCONES DAIDZEIN AND GENISTEIN IN SOYBEANS (GLYCINE MAX (L.) MERR.) USING RP HPLC METHOD

Objective: The aim of this research was develop a validation method for free aglycones analysis especially daidzein and genistein in soybean (Glycine max (L.) Merr.) using HPLC. Methods: In this study, reversed-phase HPLC (RP-HPLC) equipped with an endcapped Sun Fire TM C-18 column (150 mm x 4.6 mm, 5 μm) was used for the separation. The binary mobile phase consisted of methanol and 0.1% acetid acid (53:47). An isocratic program was used with flow rate at 1.0 mL/min and the injection volume was 10µL. The analytes were detected by using Photo-diode array (PDA) at 254 nm and the samples in various soybean varieties from Balitkabi Malang, Indonesia. Results: The developed method showed that the parameters of system suitability, selectivity, the accuracy values of recoveries for daidzein 83.0% -100.95% and genistein 80.07%-108.79%, and the precision was calculated %RSD ≤ 2% respectively, linearity of the method was the r2 values ranged from 0,9989 - 0,9991 and all the parameters the acceptable criteria of validated method. The Limit of detection (LoD) and Limit of quantitation (LoQ) indicated a good sensitive method, with LoDs values obtained were 0.05192 μg/mL and 0,0600 μg/mL for daidzein and genistein, respectively. Meanwhile, the LoQs value were 0.1731 μg/mL and 0.2000 μg/mL for daidzein and genistein, respectively. Conclusion: A simple, accurate, precise and being specific HPLC method coupled to PDA for the analysis of daidzein and genistein in various soybeans varieties was developed and validated.

Bioanalytical HPLC method development for simultaneous determination of valsartan and co-administered clopidogrel bisulfate and fenofibrate in stroke prevention in raw materials, spiked human plasma and tablets

This study reports about simple, robust and reproducible method for simultaneous bioanalytical determination of Valsartan (VAL) and co-administered Clopidogrel bisulfate (CGB) and Fenofibrate (FEN) in raw materials, spiked human plasma and tablets using isocratic RP-HPLC method. The chromatographic separation is carried out using isocratic binary mobile phase consisting of 80 mM phosphate buffer pH 3: Acetonitrile (30: 70 %; v/v) at the flow rate of 1.1 mL/min and 33 °C. A Diode array detector at wavelength 214 nm was used. Retention times for VAL, CGB and FEN were 3.1, 5.1 and 6.4 min, respectively. The calibration curves obtained were linear over the concentration ranges of 2.5 - 100 μg/mL for both VAL and CGB and 5 -100 μg/mL for FEN. The mean extraction recoveries of VAL, CGB and FEN from spiked plasma were 75.38±1.34 %, 89.91±2.17 % and 96.92±6.02 %, respectively. The limits of detection and quantification were 0.86, 0.67, 1.11 μg/mL and 2.60, 2.03, 3.36 μg/mL for VAL, CGB and FEN, respectively. The method was applied to the analysis of these drugs in spiked human plasma and in tablets as they are commonly used as a combination for prevention of stroke. Results obtained show good accuracy, precision and acceptable recoveries from plasma samples.

Validation of a UHPLC-ESI-MS/MS method for anthocyanidin quantification in potato tubers

The development of bioanalytical methods has become challenging due to sample complexity, requirements for method reliability, and speed of analysis with triple quadrupole LC-MS/MS used widely for the routine analysis of biological materials. The article presents the method development and validation results for pelargonidin and malvidin in potato tubers. The developed method uses a short C18 column, is able to measure all six common anthocyanidins, uses a binary mobile phase with acetonitrile and water both with added 1% formic acid, ESI ionisation in positive mode, 3-h hydrolysis with 2.7 M methanolic HCl at 90°C. For pelargonidin and malvidin, the method shows high recovery of 98–100%, intra-day repeatability of 6.7–17.9% (depending on the analyte and concentration level), uncertainty below 20%, and uses quadratic calibration.

Core-shell in liquid chromatography: application for determining sulphonamides in feed and meat using conventional chromatographic systems

A C18 column packed with core-shell particles was used for the chromatographic separation of sulphonamides in feed and meat by a conventional high performance liquid chromatography system coupled with a diode array detector. Two analytical methods, already used in our laboratory, have been modified without any changes in the extraction and clean-up steps and in the liquid chromatography instrumentation. Chromatographic conditions applied on a traditional 5-μm column have been optimized on a column packed with 2.6 μm core-shell particles. A binary mobile phase [acetate buffer solution at pH 4.50 and a mixture of methanol acetonitrile 50: 50 (v/v)] was employed in gradient mode at the flow rate of 1.2 mL with an injection volume of 6 μL. These chromatographic conditions allow the separation of 13 sulphonamides with an entire run of 13 minutes. Preliminary studies have been carried out comparing blanks and spiked samples of feed and meat. A good resolution and the absence of interferences were achieved in chromatograms for both matrices. Since no change was made to the sample preparation, the optimized method does not require a complete revalidation and can be used to make routine analysis faster.

Pharmacokinetic-pharmacodynamic correlation of imipenem in pediatric burn patients using a bioanalytical liquid chromatographic method

<p>A bioanalytical method was developed and applied to quantify the free imipenem concentrations for pharmacokinetics and PK/PD correlation studies of the dose adjustments required to maintain antimicrobial effectiveness in pediatric burn patients. A reverse-phase Supelcosil LC18 column (250 x 4.6 mm 5 micra), binary mobile phase consisting of 0.01 M, pH 7.0 phosphate buffer and acetonitrile (99:1, v/v), flow rate of 0.8 mL/min, was applied. The method showed good absolute recovery (above 90%), good linearity (0.25-100.0 µg/mL, r²=0.999), good sensitivity (LLOQ: 0.25 µg/mL; LLOD: 0.12 µg/mL) and acceptable stability. Inter/intraday precision values were 7.3/5.9%, and mean accuracy was 92.9%. A bioanalytical method was applied to quantify free drug concentrations in children with burns. Six pediatric burn patients (median 7.0 years old, 27.5 kg), normal renal function, and 33% total burn surface area were prospectively investigated; inhalation injuries were present in 4/6 (67%) of the patients. Plasma monitoring and PK assessments were performed using a serial blood sample collection for each set, totaling 10 sets. The PK/PD target attained (40%T>MIC) for each minimum inhibitory concentration (MIC: 0.5, 1.0, 2.0, 4.0 mg/L) occurred at a percentage higher than 80% of the sets investigated and 100% after dose adjustment. In conclusion, the purification of plasma samples using an ultrafiltration technique followed by quantification of imipenem plasma measurements using the LC method is quite simple, useful, and requires small volumes for blood sampling. In addition, a small amount of plasma (0.25 mL) is needed to guarantee drug effectiveness in pediatric burn patients. There is also a low risk of neurotoxicity, which is important because pharmacokinetics are unpredictable in these critical patients with severe hospital infection. Finally, the PK/PD target was attained for imipenem in the control of sepsis in pediatric patients with burns.</p>

Spectrophotometric and Stability–Indicating High–Performance Liquid Chromatographic Determinations of Terbutaline Sulfate

Spectrophotometric and stability-indicating HPLC procedures are described for determination of terbutaline sulfate in bulk powder and dosage form. The first procedure is based on diazo coupling of the phenolic groups of terbutaline sulfate with fast red B salt in the presence of sodium hydroxide. The colored compound developed in alkaline medium was measured at 475 nm. Different variables affecting the reaction were studied. Beer's Law is obeyed in the concentration range of 1–6 μg/mL. In the HPLC procedure, the separation was carried out on a Caltrex® AIII column, a relatively new packing material consisting of silica-bonded calix[8] arene, using an isocratic binary mobile phase, acetonitrile–ammonium acetate (50 + 50, v/v), at pH 6.2. A diode array detector was used at 280 nm. The method was validated for system suitability, linearity, precision, LOD, LOQ, specificity, stability, and robustness. The LOD and LOQ were 0.196 and 0.781 μg/mL, respectively. The recovery values of this method were between 98 and 102%, and the reproducibility was within 0.92%. Statistical comparison of the results obtained from the analysis of the studied drug to those of the official British Pharmacopoeia (2007) method using t- and F-tests showed no significant difference between them.