A new validated HPLC method for the determination of quercetin: Application to study pharmacokinetics in rats

A simple, accurate, and reproducible high‐performance liquid chromatography (HPLC) method has been developed and validated for the quantification of quercetin (QR) in rat plasma. The method involves a simple protein precipitation procedure to extract both QR and thymoquinone (TQ), the internal standard. The chromatographic analysis was achieved on a Shimadzu LC 20 A HPLC system equipped with a Supelcosil LC‐18 T C₁₈ column and an isocratic mobile phase consisting of 0.3% trichloroacetic acid in water and acetonitrile HPLC‐grade (50:50, v /v) run at a flow rate of 0.9 mL/min for 13 min. The UV detection wavelength was set at 254 nm. The method exhibited good linearity (R ² > 0.994) over the assayed concentration range (0.10–25 μg/mL) and demonstrated good intra‐day and inter‐day precision and accuracy (relative standard deviations and the deviation from predicted values were <20%). This method was also successfully applied for studying the pharmacokinetics of QR in rats following a single oral dose of QR to evaluate its pharmacokinetic parameters in rats.     

Determination of glucosamine and carisoprodol in pharmaceutical formulations by LC with pre-column derivatization and UV detection

A simple and reliable precolumn derivatization liquid chromatography method with ultraviolet detection has been developed and validated for the analysis of glucosamine (GS) in various dietary supplement formulations and raw materials. Additionally, the proposed method was used for analysis of carisoprodol (CR) found in ternary mixture with paracetamol (PR) and caffeine (CF). The linearity ranges were 1-100 μg/mL for GS, 1-150 μg/mL for CR, PR and CF. Derivatization was used with 1,2-naphthoquinone-4-sulphonic acid sodium salt in the presence of borate buffer. Chromatographic separation of GS-naphthoquinone derivative was achieved by using a mixture of acetonitrile and water (pH 7.3 adjusted with 0.1 M NaOH) in the ratio 10:90, v/v and flow-rate of 1.0 mL/min. UV detection was carried out at 280 nm. For PR, CF, and CR-naphthoquinone derivative, the chromatographic separation was achieved by using mixture of acetonitrile and 20 mM KH(2)PO(4) (pH 3.0 adjusted with phosphoric acid) in the ratio 20:80, v/v and flow-rate of 1.0 mL/min. UV detection was carried out at 275 nm. The limits of detection were 37.2, 35.9, 30.4 and 40.0 ng/mL for GS, CR, PR and CF, respectively.     

High-performance liquid chromatographic determination of pantoprazole and its main impurities in pharmaceuticals

An RP-HPLC method for simultaneous separation and quantification of pantoprazole and its five main impurities in pharmaceutical formulations was developed and validated. The separation was accomplished on a Zorbax Eclipse XDB C18 column (5 microm particle size, 150 x 4.6 mm id) using a gradient with mobile phase A [buffer-acetonitrile (70 + 30, v/v)], and mobile phase B [buffer-acetonitrile (30 + 70, v/v)]. The buffer was 0.01 M ammonium acetate solution with addition of 1 mL triethylamine/L of the solution, adjusted to pH 4.5 with orthophosphoric acid. The eluent flow rate was 1 mL/min, the temperature of the column was 30 degrees C, and the eluate was monitored at 290 nm. Linearity (r = 0.999), recovery (97.6-105.8%), RSD (0.55-1.90%), and LOQ (0.099-1.48 microg/mL) were evaluated and found to be satisfactory. The proposed method can be used for simultaneous identification and quantification of the analyzed compounds in pharmaceutical formulations.     

Quantification of arecoline (areca nut alkaloid) in neonatal biological matrices by high-performance liquid chromatography/electrospray quadrupole mass spectrometry

A high-performance liquid chromatography (HPLC) method with mass spectrometric detection is described for determination of arecoline in newborn meconium, urine and cord serum, using pilocarpine as internal standard. The analytes were extracted from neonatal biological matrices with chloroform/isopropanol (95:5, v/v) at alkaline pH. Extracts were analyzed by HPLC coupled to an electrospray (ESI) interface and a quadrupole mass spectrometer. Chromatography was performed on a C(8) reversed-phase column using 10 mM ammonium acetate (pH 4.3)/acetonitrile (90:10, v/v) as mobile phase. The mass spectrometer was operated in selected ion monitoring mode. The method was validated over the concentration range 0.005-1.00 micro g/g meconium, 0.004-1.00 micro g/mL cord serum and 0.001-1.00 micro /mL urine. Mean recoveries ranged between 86.5 and 90.7% for arecoline in the different biological matrices, with precision always better than 10%. The quantification limits of arecoline were 0.005 micro g/g meconium, 0.004 micro g/mL cord serum, and 0.001 micro g/mL urine. The method was applied to the analysis of neonatal biological matrices to assess eventual fetal exposition to arecoline. Two newborns from Asian mothers who declared areca nut consumption presented arecoline in meconium with concentrations in the range 0.006-0.008 micro g/g; also the urine from one neonate tested positive for the drug.     

Optimization of ultrasonic-assisted extraction of 3-monochloropropane-1,2-diol (MCPD) and analysis of its esters from edible oils by gas chromatography-mass spectrometry

In this paper, ultrasonic-assisted extraction of 3-chloropropane-1,2-diol and its esters from edible oils was studied with isotope dilution GC-MS. Effects of several experimental parameters, such as types and concentrations of extracting solvent, ratios of liquid to material, extraction temperature, time of ultrasonic treatment on the extraction efficiency of 3-chloropropane-1,2-diol and its esters from edible oils and sample preparation for calibration were compared and optimized. The optimal extraction conditions were suggested as 66 mg oil sample in mixture of 0.5 mL MTBE/ethyl acetate (20% v/v) and 0.5 mL of sulfuric acid/n-propanol (0.3% v/v), being extracted for 30 min at 45°C under ultrasonic irradiation. Good linearity was gained in the range of 0.020-5.000 μg/g with the limit of detection (LOD) of 0.006 μg/g (S/N = 3) and the limit of quantification (LOQ) of 0.020 μg/g (S/N = 10). The recoveries at five spiked concentrations were ranged from 91.9 to 109.3% with RSD less than 9.4%. The method was successfully applied to the determination of 3-chloropropane-1,2-diol and its esters amounts in rapeseed, sesame, peanut, camellia, and soybean oils.     

High performance liquid chromatographic method for the determination and pharmacokinetic studies of oxyresveratrol and resveratrol in rat plasma after oral administration of Smilax china extract

A sensitive and simple HPLC method has been developed and validated for the determination of oxyresveratrol (trans-2,4,3',5'-tetrahydroxystilbene, OXY) and resveratrol (trans-3,5,4'-trihydroxystilbene, RES) in rat plasma. The plasma samples were extracted with ethyl acetate and analyzed using HPLC on an Aglient Zorbax SB-C(18) column (250 x 4.6 mm, 5 microm) at a wavelength 320 nm, with a linear gradient of (A) acetonitrile and (B) 0.5% aqueous acetic acid (v/v), at a flow rate of 1.0 mL/min. The method was linear over the range of 0.1265-25.3 microg/mL for OXY and 0.117-23.4 microg/mL for RES. The extraction recovery for OXY, RES and internal standard ranged from 71.1 to 88.3%. The intra- and inter-day precisions were better than 10%, and the accuracy ranged from 89 to 108%. The validated method was used to study the pharmacokinetic profiles of OXY and RES in rat plasma after oral administration of Smilax china root extract.     

Biological assay and liquid chromatographic method for analysis of moxifloxacin in tablets

A microbiological assay and a liquid chromatographic method were validated for quantitation of moxifloxacin in tablets. The microbiological method consisted of a cylinder-plate agar diffusion assay using Micrococcus luteus ATCC 9341 as the test microorganism and phosphate buffer (0.1M, pH 8.0) as the diluent solution. The response graphs for standard and sample solutions were linear (r = 0.9479), and no parallelism deviations were detected in the tested levels of concentration (4.0, 8.0, and 16.0 microg/mL). The interday precision was 2.73%. Recovery values were between 96.25 and 100.5%. The chromatographic analyses were performed using a Shim-pack CLC-ODS column (250 x 4.6 mm, 5 microm) with a mobile phase consisting of (A) a mixture of phosphoric acid (0.17%, v/v) with tetramethylammonium hydroxide (0.05M) and acetonitrile (95 + 5, v/v) and (B) methanol (55 + 45, v/v) adjusted to pH 3.0. The flow rate was 1.0 mL/min, and detection was made at 294 nm. The method was linear in a range from 12.0 to 42 microg/mL (r = 0.9999), and the interday precision was 1.39%. Recovery ranged between 101.9 and 103.81%. Both validated methods were used to quantify the moxifloxacin content in tablets exposed to ultraviolet radiation, and similar results were obtained.     

Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.     

High performance liquid chromatographic analysis of dehydroepiandrosterone and its pharmaceutical tablet formulation

A rapid, sensitive and stability indicating high performance liquid chromatographic method was developed and validated for the analysis of dehydroepiandrosterone (DHEA) in pharmaceutical tablet formulation. The analysis was done on a Supelcosil C(18) column (25 cm x 4.6 mm i.d., 5 microm). The mobile phase consisted of methanol:sodium acetate buffer solution (5 g/L):acetic acid (500 mL/L), 57:42:1, v/v/v, adjusted to pH 5 at a flow rate of 1 mL/min. Detection was carried out at a wavelength of 258 nm. The polynomial regression data for the calibration curve showed good linear relationship in the concentration range of 0.2-1 mg/mL with r = 0.9996. The method was validated for precision, accuracy and recovery. The limit of detection was found to be 50 ng/ microL. The method was applied for the analysis of DHEA in its pharmaceutical tablet formulation. The effects of different buffers and alcohols on the retention of DHEA were studied and the role of acetic acid as an organic phase modifier was also investigated.     

Liquid chromatographic high-throughput analysis of the new ultra-short acting hypnotic 'HIE-124' and its metabolite in mice serum using a monolithic silica column

For the first time, a fast, high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of the new ultra-short hypnotic HIE-124 and its metabolite in mice serum. Each compound, together with carbamazepine (internal standard) was extracted from the serum matrix using liquid-liquid extraction (LLE). Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column (100 mm × 4.6 mm i.d.) under isocratic conditions using a mobile phase of 65:35 (v/v), 20 mM phosphate buffer (pH 7.0 adjusted with phosphoric acid)-acetonitrile. The elution of the analytes were monitored at 240 nm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 2.5 mL min(-1). The total run time of the assay was 2 min. The method was validated over the range of 60-2000 ng mL(-1) for HIE-124 and 200-1600 ng mL(-1) for the metabolite (r(2) = 0.99). The limit of detection (LOD) for HIE-124 and its metabolite were 20 ng mL(-1) and 65 ng mL(-1), respectively. The proposed method was validated in compliance with ICH guidelines, in terms of accuracy, precision, limits of detection and quantitation and other aspects of analytical validation. The developed method could be used for the trace analyses of HIE-124 and its metabolite in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum.     

Determination of amlodipine in pharmaceutical dosage forms by liquid chromatography and ultraviolet spectrophotometry

A liquid chromatography (LC) method and an ultraviolet (UV) spectrophotometric method were developed and validated for quantitative determination of amlodipine in tablets and compounded capsules. The isocratic LC analyses were performed on an RP18 column using a mobile phase composed of 0.1% (v/v) ortho-phosphoric acid (pH 3.0) -acetonitrile (60 + 40, v/v) at a flow rate of 1.0 mL/min. The UV spectrophotometric method was performed at 238 nm. The analytical methods were validated according to International Conference on Harmonization Guidelines. The calibration graphs were linear [correlation coefficient (r) > 0.999] in the studied concentration range of 10-30 microg/mL for LC and 10-35 microg/mL for UV spectrophotometry. The relative standard deviation values for intraday and interday precision studies were less than 2%, and the accuracy was greater than 98% for both methods. The specificity of the LC method was proved using forced degradation. Statistical analyses showed no significant difference between the results obtained by the 2 methods. The proposed methods are precise and accurate and can be applied directly and easily to the oral pharmaceutical preparations of amlodipine.     

Determination of omeprazole in bulk and injectable preparations by liquid chromatography

An accurate, simple, reproducible, and sensitive liquid chromatographic method was developed and validated for the determination of omeprazole in powder for injection and in pellets. The analyses were performed at room temperature on a reversed-phase C18 column of 250 x 4.6 mm id, 5 microm particle size. The mobile phase, composed of methanol-water (90 + 10, v/v), was pumped at a constant flow rate of 1.5 mL/min. Detection was performed on a UV detector at 301 nm. The method was validated in terms of linearity, precision, accuracy, and ruggedness. The response was linear in the range 32-48 microg/mL (r2 = 0.9976). The relative standard deviation values for intra- and interday precision studies were 1.22 and 1.56% for injectable and 2.13 and 2.45% for pellets, respectively. Recoveries ranged between 95.81 and 100.48%.     

Stability-indicating methods for determination of tiapride in pure form, pharmaceutical preparation, and human plasma

Four different stability-indicating procedures are described for determination of tiapride in pure form, dosage form, and human plasma. Second derivative (D2), first derivative of ratio spectra (1DD), spectrofluorimetric, and high-performance column liquid chromatographic (LC) methods are proposed for determination of tiapride in presence of its acid-induced degradation products, namely 2-methoxy-5-(methylsulfonyl) benzoic acid and 2-diethylaminoethylamine. These approaches were successfully applied to quantify tiapride using the information included in the absorption, excitation, and emission spectra of the appropriate solutions. In the D2 method, Beer's law was obeyed in the concentration range of 1.5-9 microg/mL with a mean recovery of 99.94 +/- 1.38% at 253.4 nm using absolute ethanol as a solvent. In 1DD, which is based on the simultaneous use of the first derivative of ratio spectra and measurement at 245 nm in absolute ethanolic solution, Beer's law was obeyed over a concentration range of 1.5-9 microg/mL with mean recovery 99.64 +/- 1.08%. The spectrofluorimetric method is based on the determination of tiapride native fluorescence at 339 nm emission wavelength and 230 nm excitation wavelength using water-methanol (8 + 2, v/v). The calibration curve was linear over the range of 0.2-3 microg/mL with mean recovery of 99.66 +/- 1.46%. This method was also applied for determination of tiapride in human plasma. A reversed-phase LC method performed at ambient temperature was validated for determination of tiapride using methanol-deionized water-triethylamine (107 + 93 + 0.16, v/v/v) as the mobile phase. Sulpiride was used as an internal standard at a flow rate of 1 mL/min with ultraviolet detection at 214 nm. A linear relation was obtained over a concentration range of 2-30 microg/mL with mean recovery of 99.66 +/- 0.9%. Results were statistically analyzed and compared with those obtained by applying the reference method. They proved both accuracy and precision.     

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.     

Simultaneous determination of leflunomide and its active metabolite,A77 1726, in human plasma by high-performance liquid chromatography

The isoxazol derivative leflunomide [N-(4'-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide] is an inhibitor of de novo pyrimidine synthesis used for the treatment of rheumatoid artrithis. In the present study, a liquid-liquid extraction-based reversed-phase HPLC method with UV detection was validated and applied for the analysis of leflunomide and its active metabolite, A77 1726, in human plasma. The analytes were separated using a mobile phase, consisting of acetonitrile, water and formic acid (40/59.8/0.2, v/v), at a flow rate of 0.5 mL/min, and UV detection at 261 nm. The retention times for A77 1726, leflunomide and warfarin (internal standard) were 8.2, 16.2 and 12.2 min, respectively. The validated quantification range of the method was 0.05-100 micro g/mL for leflunomide and 0.1-100 micro g/mL for A77 1726. The developed procedure was applied to assess steady-state plasma concentrations of A77 1726 in patients with rheumatoid arthritis treated with 10 or 20 mg leflunomide per day.     

反相高效液相色谱法同时测定片剂中的加替沙星与盐酸氨溴索

 A reversed-phase high performance liquid chromatography (HPLC) method was developed, validated, and used for the quantitative determination of gatifloxacin (GA) and ambroxol hydrochloride (AM), from its tablet dosage form. Chromatographic separation was performed on a HiQ Sil C18 column (250 mm×4.6 mm, 5 μm), with a mobile phase comprising of a mixture of 0.01 mol/L potassium dihydrogen orthophosphate buffer and acetonitrile (70∶30, v/v), and pH adjusted to 3 with orthophosphoric acid, at a flow rate of 1 mL/min, with detection at 247 nm. Separation was completed in less than 10 min. As per International Conference on Harmonisation (ICH) guidelines the method was validated for linearity, accuracy, precision, limit of quantitation, limit of detection, and robustness. Linearity of GA was found to be in the range of 10-60 μg/mL and that for AM was found to be 5-30 μg/mL. The correlation coefficients were 0.9996 and 0.9993 for GA and AM respectively. The results of the tablet analysis (n=5) were found to be 99.94% with ±0.25% standard deviation (SD) and 99.98% with±0.36% SD for GA and AM respectively. Percent recovery of GA was found to be 99.92%-100.02% and that of AM was 99.86%-100.16%. The assay experiment shows that the method is free from interference of excipients. This demonstrates that the developed HPLC method is simple, linear, precise, and accurate, and can be conveniently adopted for the routine quality control analysis of the tablet.     

Stability indicating reversed-phase high-performance liquid chromatographic and thin layer densitometric methods for the determination of ziprasidone in bulk powder and in pharmaceutical formulations

Two sensitive and reproducible methods were developed and validated for the determination of ziprasidone (ZIP) in the presence of its degradation products in pure form and in pharmaceutical formulations. The fi rst method was based on reversed-phase high-performance liquid chromatography (HPLC), on a Lichrosorb RP C(18) column using water:acetonitrile:phosphoric acid (76:24:0.5 v/v/v) as the mobile phase at a fl ow rate of 1.5 mL min(-1) at ambient temperature. Quantification was achieved with UV detection at 229 nm over a concentration range of 10-500 micro g mL(-1) with mean percentage recovery of 99.71 +/- 0.55. The method retained its accuracy in presence of up to 90% of ZIP degradation products. The second method was based on TLC separation of ZIP from its degradation products followed by densitometric measurement of the intact drug spot at 247 nm. The separation was carried out on aluminium sheet of silica gel 60 F(254) using choloroform:methanol:glacial acetic acid (75:5:4.5 v/v/v) as the mobile phase, over a concentration range of 1-10 micro g per spot and mean percentage recovery of 99.26 +/- 0.39. Both methods were applied successfully to laboratory prepared mixtures and pharmaceutical capsules.     

Development and validation of a rapid and efficient method for simultaneous determination of methylxanthines and their metabolites in urine using monolithic HPLC columns

A new rapid, sensitive and validated HPLC method has been developed for the determination of methylxanthines and their metabolites in asthmatic patients. The method was initiated by using spiked urine samples on a silica monolithic column as a novel packing material. The mobile phase consisted of 10 mM potassium dihydrogen phosphate buffer/methanol (87.5:12.5 v/v), at a flow rate 1 mL/min. Detection was set at 274 nm. The LOQ for all the compounds ranged from 14 to 41 ng/mL. Excellent linearity was achieved over the studied range of concentration with correlation coefficients 0.9991–0.9998 (n = 6). The developed method was validated by precision and accuracy with RSD <2.55%. On extraction of the drugs and metabolites from the urine samples high recoveries were achieved ranging from 82.06 to 98.34% w/w on RP18 cartridges and methanol/chloroform (20:80 v/v) as the extraction solvent. This method has advantages over other methods using conventional C18 packings.     

HPLC analysis and pharmacokinetic study of quercitrin and isoquercitrin in rat plasma after administration of Hypericum japonicum thunb. extract

A simple HPLC method was developed for determination of quercitrin and isoquercitrin in rat plasma. Reversed-phase HPLC was employed for the quantitative analysis using kaempferol-3-O-beta-D-glucopyranoside-7-O-alpha-L-rhamnoside as an internal standard. Following extraction from the plasma samples with ethyl acetate-isopropanol (95:5, v/v), these two compounds were successfully separated on a Luna C(18) column (250 x 4.6 mm, 5 microm) with isocratic elution of acetonitrile-0.5% aqueous acetic acid (17:83, v/v) as the mobile phase. The flow-rate was set at 1 mL/min and the eluent was detected at 350 nm for both quercitrin and isoquercitrin. The method was linear over the studied ranges of 50-6000 and 50-5000 ng/mL for quercitrin and isoquercitrin, respectively. The intra- and inter-day precisions of the analysis were better than 13.1 and 13.2%, respectively. The lower limits of quantitation for quercitrin and isoquercitrin in plasma were both of 50 ng/mL. The mean extraction recoveries were 73 and 61% for quercitrin and isoquercitrin, respectively. The validated method was successfully applied to pharmacokinetic studies of the two analytes in rat plasma after the oral administration of Hypericum japonicum thunb. ethanol extract.     

Simple determination of pirfenidone in rat plasma via high-performance liquid chromatography

A simple, rapid and reliable high-performance liquid chromatographic method was developed and validated for the determination of pirfenidone and its major metabolites in rat plasma. Plasma proteins were precipitated with perchloric acid (10%, v/v) and the supernatant after centrifugation was determined using high-performance liquid chromatography. The analysis was carried out on a Lichrospher C(18) column (250 x 4.6 mm i.d., 5 microm). The mobile phase consisted of acetonitrile-water containing 0.2% acetic acid (23:77, v/v) at a flow-rate of 1 mL/min. The eluant was detected at 310 nm. The calibration curves were linear over a concentration range from 0.15 to 76.67 microg/mL. The accuracy (relative error) of the assay ranged from -2.6 to 7.9% and the precision (coefficient of variation) was less than 4.5%. The established method has been successfully applied to a pharmacokinetic study of pirfenidone following a single oral dose to rats.