Development and full validation of an HPLC methodology to quantify atorvastatin and curcumin after their intranasal co‐delivery to mice

There is increasing interest in atorvastatin and curcumin owing to their potential anticancer activity. A new, accurate and sensitive HPLC method was developed, for the first time, to simultaneously quantify atorvastatin and curcumin in mouse plasma and brain, liver, lung and spleen tissues following protein precipitation sample preparation. The chromatographic separation was achieved in 13 min on a C₁₈ column, at 35°C, using a mobile phase composed of acetonitrile–methanol–2% (v/v) acetic acid (37.5:2.5:60, v/v/v) at a flow rate of 1.0 mL/min. The detection of analytes and internal standard was carried out at 247, 425 and 250 nm, respectively. According to international guidelines, the method was shown to be selective, with lower limits of quantification ranging from 10 to 500 ng/mL for curcumin, and from 100 to 600 ng/mL for atorvastatin, linear over a wide concentration range (r² ≥ 0.9971) and with acceptable accuracy (bias ± 12.29%) and precision (coefficient of variation ≤13.15%). The analytes were reproducibly recovered at a percentage >81.10% and demonstrated to be stable under various experimental conditions in all biological matrices. This method can be easily applied to in vivo biodistribution studies related to the intranasal administration of atorvastatin and curcumin, separately or simultaneously.     

On-line solid-phase extraction-HPLC-fluorescence detection for simultaneous determination of puerarin and daidzein in human serum

Response surface methodology (RSM) was applied to the optimization of on-line solid-phase extraction (SPE) parameters, and an automated system of on-line SPE coupled with high-performance liquid chromatography (HPLC) with fluorescence detection was developed for the determination of puerarin and daidzein in human serum. The human serum sample of 50 μL was injected into a conditioned C18 SPE cartridge, and the matrix was washed out with acetonitrile-KH₂PO₄-triethylamine buffer (0.01 M, pH 7.4) (3:97, v/v) for 3 min at a flow rate of 0.25 mL/min. Then the target analytes were eluted and transferred to the analytical column. A chromatographic gradient elution was programmed with the mobile phase consisting of acetonitrile and KH₂PO₄-triethylamine buffer, and the analytes were determined with a fluorescence detector at excitation wavelength of 350 nm and emission wavelength of 472 nm, respectively. The proposed method presented good linear relations (0.85-170 μg/mL for puerarin and 0.2-40 μg/mL for daidzein), satisfactory precision (RSD < 8%), and accredited recovery (92.5-107.8%).     

Development and validation of a fast isocratic liquid chromatography method for the simultaneous determination of norfloxacin,lomefloxacin and ciprofloxacin in human plasma

A simple and fast liquid chromatographic method coupled with fluorescence detection (LC‐FD) is reported, for the first time, for the simultaneous quantification of norfloxacin (NOR), ciprofloxacin (CIP) and lomefloxacin (LOM) in human plasma, using levofloxacin as internal standard (IS). Sample preparation consists of a single‐step precipitation of plasma proteins followed by vortex‐mixing and centrifugation. Chromatographic separation was achieved within 7 min on a reversed‐phase C₁₈ column with a mobile phase consisting of 0.1% aqueous formic acid (pH = 3.0, triethylamine)–methanol (82:18, v/v) pumped isocratically at 1.2 mL/min. The detector was set at excitation/emission wavelengths of 278/450 nm. Calibration curves were linear (r² ≥ 0.994) in the range of 0.02–5.0 µg/mL, and the limit of quantification was established at 0.02 µg/mL for all analytes (NOR, CIP and LOM). The overall precision did not exceed 8.19% and accuracy was within ±10.91%. NOR, CIP and LOM were extracted from human plasma with an overall mean recovery ranged from 90.1 to 111.5%. No interferences were observed at the retention times of the analytes and IS. This novel LC‐FD method enables the reliable determination of NOR, CIP and LOM in a single chromatographic run, which may be suitable to support human pharmacokinetic‐based studies with those antimicrobial agents. Copyright © 2010 John Wiley & Sons, Ltd.     

Reversed-Phase High-Performance Liquid Chromatographic Determination of Nicotine in Pesticide Formulations

Abstract

A simple and rapid reversed-phase high-performance liquid chromatographic procedure is described for the determination of nicotine in liquid formulations. Samples are diluted with methanol, and naphthalene is added as the internal standard. Peak height ratios obtained from injections of standard and sample filtrates are used for quanti-tation. An eluting solvent of 0.05M (NH₄)₂ HPO4 (pH 7.5) -methanol (40/60, v/v) at a flow of 2 mL/min gives retention times of 3.13 and 6.88 min respectively for nicotine and naphthalene. Sample analysis can be completed in approximately one hour by the method described as compared to 1.5 days required by the Official AOAC gravimetric method (6.176–6.177).     

A sensitive LC‐MS/MS method for the quantification of febuxostat in human plasma and its pharmacokinetic application

An improved, simple and highly sensitive LC‐MS/MS method has been developed and validated for quantification of febuxostat with 100 μL human plasma using febuxostat‐d₇ as an internal standard (IS) according to regulatory guidelines. The analyte and IS were extracted from human plasma via liquid–liquid extraction using diethyl ether. The chromatographic separation was achieved on a Zorbax C₁₈ column using a mixture of acetonitrile and 5 mm ammonium formate (60:40, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The total run time was 5.0 min and the elution of febuxostat and IS occurred at 1.0 and 1.5 min, respectively. A linear response function was established for the range of concentrations 1–6000 ng/mL (r > 0.99). The precursor to product ion transitions monitored for febuxostat and IS were m/z 317.1 → 261.1 and 324.2 → 262.1, respectively. The intra‐ and inter‐day precisions (%RSD) were within 1.29–9.19 and 2.85–7.69%, respectively. The proposed method was successfully applied to pharmacokinetic studies in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitation of unbound sunitinib and its metabolite N-desethyl sunitinib (SU12662) in human plasma by equilibrium dialysis and liquid chromatography-tandem mass spectrometry: application to a pharmacokinetic study

A rapid, selective, and sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of unbound sunitinib and its active metabolite N‐desethyl sunitinib in plasma. Plasma and post‐dialysis buffer samples were extracted using a liquid–liquid extraction procedure with acetonitrile–n‐butylchloride (1:4, v/v). Chromatographic separation was achieved on a Waters X‐Terra® MS RP₁₈ column with a mobile phase consisting of acetonitrile and water (60:40, v/v) containing formic acid (0.1%, v/v) using an isocratic run, at a flow‐rate of 0.2 mL/min. Analytes were detected by electrospray tandem mass spectrometry in the selective reaction monitoring mode. Linear calibration curves were generated over the ranges 0.1–100 and 0.02–5 ng/mL for sunitinib and 0.2–200 and 0.04–10 ng/mL for N‐desethyl sunitinib in plasma and in phosphate‐buffered solution, respectively. The values for both within‐day and between‐day precision and accuracy were well within the generally accepted criteria for analytical methods. The analytical range was sufficient to determine the unbound and total concentrations of both analytes. The method was applied for measurement unbound concentrations in addition to total concentrations of sunitinib and its metabolite in plasma of a cancer patient receiving 50 mg daily dose. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous quantification of cefpirome and cetirizine or levocetirizine in pharmaceutical formulations and human plasma by RP-HPLC

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the simultaneous determination and quantification of cefpirome and cetirizine or cefpirome and levocetirizine in pharmaceutical formulations and human plasma without changing the chromatographic conditions is described. Chromatographic separations were performed on a prepacked Nucleosil 120, C₁₈ (5 μm, 12.5 ± 0.46 mm) column using CH₃CN: H₂O (75: 25, v/v) as a mobile phase at a flow rate of 1 mL/min while UV detection was performed at 232 nm for monitoring the effluent. A number of other brands of C₁₈ columns were also employed which had a significant effect on the separation. The method has been validated over the concentration range of 0.5–50 μg/mL (r ₂ > 0.999). The limit of detection (LOD) and quantification (LOQ) for cefpirome and levocetirzine in pharmaceutical formulations and serum were in the range 0.24–1.31 μg/mL. Analytical recovery from human plasma was >98%, and the within and between-day relative standard deviation was <3.1%. The small sample volume and simplicity of preparation make this method suitable for use in pharmaceutical industries, drug research centers, clinical laboratories, and forensic medical centers. The text was submitted by the authors in English.     

Simultaneous determination of phenytoin and dextromethorphan in urine by solid-phase extraction and HPLC-DAD

A rapid and simple high-performance liquid chromatographic method with photodiode array detection was developed for the separation and the simultaneous determination of phenytoin and dextromethorphan in human urine. Analysis was performed in less than 4.5 min in isocratic mode on a reversed-phase C18 column (5 microm; 150 x 4.6 mm) using a mobile phase composed of acetonitrile-buffer phosphate 0.01 M (60:40, v/v) adjusted to pH 6.0, at 1 mL/min flow rate and UV absorbance at 210 nm. The elution order of analytes was dextromethorphan (DXM), Internal Standard (IS), and phenytoin (PHT). Calibration curves were linear in the 7.5-25 microg/mL range for PHT and in the 10-30 microg/mL range for DXM. Spike recoveries for urine samples prepared at three spiking levels ranged from 97.8 to 102.3% for PHT and from 94.8 to 100.4% for DXM. The detection limit (LOD) values ranged from 0.08 microg/mL for PHT to 0.5 microg/mL for DXM. The quantitation limit (LOQ) values ranged from 0.3 microg/mL for PHT to 1.6 microg/mL for DXM. The sample preparation method involves a rapid and simple procedure based on solid-phase extraction using a C18 reversed-phase column. Validation of the optimised method was carried out according to the ICH guidelines. The method developed in this study allows the reliable simultaneous analysis of PHT and DXM, drugs that were never quantified together in previously reported analytical methods. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human urine of epileptic patients.     

A sensitive LC–MS/MS‐based bioanalytical method for quantification of salviaflaside and rosmarinic acid in rat plasma and its application in a pharmacokinetic study

A selective and sensitive liquid chromatography tandem mass spectrometry method was developed for the simultaneous determination of salviaflaside and rosmarinic acid in rat plasma. Sample preparation was carried out through liquid–liquid extraction with ethyl acetate using curculigoside as internal standard (IS). The analytes were determined by selected reaction monitoring operated in the positive ESI mode. Chromatographic separation was performed on an Agilent Eclipse Plus C₁₈ column (100 × 4.6 mm, 1.8 μm) with a mobile phase consisting of methanol–water–formic acid (50:50:0.1, v/v/v) at a flow rate of 0.3 mL/min. The run time was 1.9 min per sample and the injection volume was 5 μL. The method had an LLOQ of 1.6 ng/mL for salviaflaside and 0.94 ng/mL for rosmarinic acid in plasma. The linear calibration curves were fitted over the range of 1.6–320 ng/mL for salviaflaside and 0.94–188 ng/mL for rosmarinic acid in plasma with correlation coefficients (r²) >0.99. Intra‐ and inter‐day precisions (relative standard deviation) were < 13.5%, and accuracies (relative error) were between −8.6% and 14.5% for all quality control samples. The method was validated and applied to the pharmacokinetics of salviaflaside and rosmarinic acid in plasma after oral administration of Prunella vulgaris extract to rats.     

Simultaneous determination of chlorzoxazone and ketoprofen in binary mixtures and in ternary mixtures containing the chlorzoxazone degradation product by reversed-phase liquid chromatography

New, simple, rapid, and precise reversed-phase high-performance liquid chromatographic (LC) methods were developed for the simultaneous determination of chlorzoxazone (CH) and ketoprofen (KT) in binary mixtures and in ternary mixtures containing the CH degradation product, 2-amino-4-chlorophenol (CD). The analytes were separated by LC on a Lichrosphere 60 C18 column (250 x 4 mm, 5 microm). The mobile phases, methanol-water (40:60, v/v) at 1 mL/min and methanol-0.05% phosphoric acid (60:40, v/v, pH 2.81) at 1.5 mL/min, satisfactorily resolved the binary and ternary mixtures, respectively. The UV detector was operated at 280 nm for the determination of CH and at 254 nm for the determination of KT and CD. Linearity, accuracy, and precision were found to be acceptable over the concentration ranges of 20-240 and 5-60 microg/mL for CH and KT, respectively, in the binary mixtures and 50-300, 10-60, and 20-160 microg/mL for CH, KT, and CD, respectively, in the ternary mixtures. The optimized methods proved to be specific, robust, and accurate for the quality control of CH and KT in pharmaceutical preparations.     

Simultaneous determination of artificial sweeteners, preservatives, caffeine, theobromine and theophylline in food and pharmaceutical preparations by ion chromatography.

A novel ion chromatographic method was proposed for the simultaneous determination of artificial sweeteners (sodium saccharin, aspartame, acesulfame-K), preservatives (benzoic acid, sorbic acid), caffeine, theobromine and theophylline. The separation was performed on an anion-exchange analytical column operated at 40 degrees C within 45 min by an isocratic elution with 5 mM aqueous NaH2PO4 (pH 8.20) solution containing 4% (v/v) acetonitrile as eluent, and the determination by wavelength-switching ultraviolet absorbance detection. The detection limits (signal-to-noise ratio 3:1) for all analytes were below the sub-microg/ml level. Under the experimental conditions, several organic acids, including citric acid, malic acid, tartaric acid and ascorbic acid, did not interfere with the determination. The method has been successfully applied to the analysis of various food and pharmaceutical preparations, and the average recoveries for real samples ranged from 85 to 104%. The levels of all analytes determined by this method were in good agreement with those obtained by the high-performance liquid chromatographic procedure. The results also indicated that ion chromatography would be possibly a beneficial alternative to conventional high-performance liquid chromatography for the separation and determination of these compounds.     

Development of a tandem thin-layer chromatography-high-performance liquid chromatography method for the identification and determination of corticosteroids in cosmetic products

A solid-phase extraction clean-up and and a liquid chromatographic method with ultraviolet detection were developed for the analysis of 51 corticosteroids in cosmetic samples in order to screen commercial samples for the presence of undeclared synthetic corticosteroids. A thin-layer chromatographic analysis was carried out on silica gel plates, using different eluants and detection reagents. When such a preliminary chromatographic separation gave some indications about the presence of steroid compounds, the methanol extracts from real samples were applied to a solid-phase extraction C₁₈ cartridge, and the analytes eluted with ethyl ether. The high-performance liquid chromatographic separation was then carried out for the identification and determination of the analytes using a Purospher RP-18 column, an isocratic or a gradient elution with a mixture acetonitrile-water and a photodiode-array detector. The accuracy of the method was determined by spiking experiments on home-made cosmetic samples. The analytical recoveries were satisfactory.     

Analysis of Temocillin and Impurities by Reversed Phase Liquid Chromatography: Development and Validation of the Method

A robust, specific, precise and sensitive high-performance liquid chromatographic method has been described for purity control of temocillin. Chromatographic separation was achieved using a Symmetry C₁₈ (150 × 4.6 mm, 5 µm) column kept at 30 °C. The mobile phase consisted of a gradient mixture of mobile phases A (5 g/L solution of Na₂HPO₄·2H₂O, pH 7) and B (ACN-MeOH-H₂O, 50:10:40 v/v/v) pumped at a flow rate of 1.0 mL/min. UV detection was performed at 235 nm. The developed method was validated according to the ICH guidelines for its robustness, selectivity, sensitivity, precision and linearity. An experimental design was applied for the robustness study. Linearity was assessed both at impurity level in the range from LOQ to 10 % and assay level from 25 % to 150 % (0.6 mg/mL = 100 %). It is the first liquid chromatographic method described for the separation of temocillin and its potential impurities. It was possible to identify four degradation products from the forced degradation studies. The degradants do not interfere with the main peak and other known impurities showing that the method is specific and stability-indicating.     

A comparative study of the separation of oleanolic acid and ursolic acid in Prunella vulgaris by high-performance liquid chromatography and cyclodextrin-modified micellar electrokinetic chromatography

A high-performance liquid chromatographic (HPLC) method and a cyclodextrin-modified micellar electrokinetic chromatographic (CD-MEKC) method were developed to separate and determine oleanolic acid (OA) and ursolic acid (UA) in Prunella vulgaris. HPLC separations were carried out on a Hedera ODS C18 column with methanol -H₂O- acetic acid (85:15:0.3, v/v/v) as mobile phase at a flow-rate of 0.8 ml min^?1. CD-MEKC analysis was performed on a CL1030 capillary electrophoresis system with a 6% (v/v) methanol solution (pH = 9.0) containing 10 mM disodium tetraborate, 10 mM sodium dihydrogen phosphate, 50 mM sodium dodecylsulfate (SDS), 15 mM 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD) as background electrolyte. The analytical results of HPLC and CD-MEKC were compared with each other. CD-MEKC has better analytical efficiency for two components, and the analytical time (15 min) was shorter than that of HPLC (35 min).     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

固相萃取净化及超高效液相色谱-串联质谱法测定橡胶制品中的13种N-亚硝胺

建立了固相萃取(SPE)净化、超高效液相色谱-串联质谱(UHPLC-MS/MS)测定橡胶制品中13种N-亚硝胺的方法。样品于密闭萃取瓶中于60 ℃下用甲醇超声萃取30 min,C₁₈固相萃取小柱对萃取液进行净化,经C₁₈色谱柱分离,最后用电喷雾正离子(ESI⁺)和多重反应监测模式(MRM)对13种N-亚硝胺进行定性、定量测定。实验中对样品前处理、色谱分离条件和质谱检测条件进行了优化。在优化的实验条件下,橡胶样品中添加N-亚硝基二甲胺(NDMA)与N-亚硝基-二乙基胺(NDEA)为500 μg/kg、其他组分均为50 μg/kg时,各组分的相对标准偏差(RSD,n=7)小于10%;在实际样品中的加标回收率为70.7%~117.0%;方法的检出限(LOD,以10倍标准偏差计)为0.5~500 μg/kg。方法可应用于橡胶制品中13种N-亚硝胺的测定。     

Simultaneous determination of ten constituents in herbal formulation Sipjeondaebo-tang using HPLC-PDA

A simple, rapid, and accurate high-performance liquid chromatographic method was applied to the quantitative determination of 10 components, namely, 5-hydroxymethyl-2-furaldehyde, albiflorin, paeoniflorin, liquiritin, ferulic acid, nodakenin, coumarin, cinnamic acid, cinnamaldehyde and glycyrrhizin in the traditional herbal formulation Sipjeondaebo-tang (SJDBT). All components were separated within 40 min using a Gemini C₁₈ column maintained at 40°C. The mobile phase was composed of 1.0% (v/v) aqueous acetic acid and 1.0% (v/v) acetic acid in acetonitrile by gradient elution. The flow rate was 1.0 mL/min and the detector was a photodiode array (PDA) set at 230, 254, 280, 320, and 330 nm. The calibration curves showed good linearity (r ² ≥ 0.9993) in different concentration ranges. The recovery of each component was in the range of 92.3–104.8%, with relative standard deviations (RSDs) less than 2.5%. The RSDs for intra- and inter-day precision were 0.09–2.8% and 0.07–3.2%, respectively. The amounts of the 10 components in SJDBT were in the range of 0.18–5.77 mg/g.     

Simultaneous quantitative determination of resorcinol and 1-Naphthol in haircolor products by high-performance liquid chromatography

Summary A high-performance liquid chromatographic method has been developed for the simultaneous quantitative determination of resorcinol and 1-naphthol in hair tonic and haircolor products. The two compounds could be separated on a μBondapak C₁₈ column by elution with 50∶50 (v/v) methanol-water as mobile phase. The retention times of resorcinol and 1-naphthol were 2.5 and 7.0 min, respectively. Seven dye intermediates could be analyzed within 12 min without any interference with the peaks of resorcinol and 1-naphthol from other cosmetic ingredients present in the haircolor liquid or cream. The results obtained were in good agreement with those obtained by a spectrophotometric method.     

Comparative methodology in the determination of alpha-oxocarboxylates in aqueous solution ion chromatography versus gas chromatography after oximation, extraction and esterification

A direct, simple and rapid high-performance liquid chromatographic method has been developed for the determination of ketoprofen with ibuprofen as internal standard. Samples were chromatographed on a 5 μm Kromasil 100 C₁₈ column. The mobile phase was a mixture of acetonitrile–0.01 M KH₂PO₄ adjusted to pH 1.5 with orthophosphoric acid 85% (60:40, v/v). Detection was at 260 nm and the run time was 10 min. The detector response was found to be linear in the concentration range 0.02 to 40 μg/ml. This HPLC assay has been applied to measure the “in vitro” percutaneous penetration of ketoprofen through rat skin.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.