Multivariate optimization of a headspace solid‐phase microextraction method followed by gas chromatography with mass spectrometry for the determination of terpenes in Nicotiana langsdorffii

A simple and sensitive procedure based on headspace solid‐phase microextraction and gas chromatography with mass spectrometry was developed for the determination of five terpenes (α‐pinene, limonene, linalool, α‐terpineol, and geraniol) in the leaves of Nicotiana langsdorffii. The microextraction conditions (extraction temperature, equilibration time, and extraction time) were optimized by means of a Doehlert design. The experimental design showed that, for α‐pinene and limonene, a low temperature and a long extraction time were needed for optimal extraction, while linalool, α‐terpineol, and geraniol required a high temperature and a long extraction time. The chosen compromise conditions were temperature 60°C, equilibration time 15 min and extraction time 50 min. The main analytical figures of the optimized method were evaluated; LODs ranged from 0.07 ng/g (α‐pinene) to 8.0 ng/g (geraniol), while intraday and interday repeatability were in the range 10–17% and 9–13%, respectively. Finally, the procedure was applied to in vitro wild‐type and transgenic specimens of N. langsdorffii subjected to abiotic stresses (chemical and heat stress). With the exception of geraniol (75–374 ng/g), low concentration levels of terpenes were measured (ng/g level or lower); some interesting variations in terpene concentration induced by abiotic stress were observed.     

Simultaneous determination of hydrochlorothiazide and losartan potassium in tablets by high-performance low-pressure chromatography using a multi-syringe burette coupled to a monolithic column

This contribution describes use of a separation method based on on-line coupling of a multisyringe flow system with a chromatographic monolithic column for simultaneous determination of hydrochlorothiazide and losartan potassium in tablets. The system comprised a multisyringe module, three low-pressure solenoid valves, a monolithic C₁₈ column (25 mm × 4.6 mm i.d.), and a diode-array detector. The mobile phase was 10 mmol L⁻¹ potassium dihydrogen phosphate (pH 3.1)-acetonitrile-methanol (65:33:2 v/v/v) at a flow rate 0.8 mL min⁻¹. UV detection was carried out at 226 nm. The multi-syringe chromatographic (MSC) method with UV spectrophotometric detection was optimized and validated. Results from validation were very good. The analysis time was about 400 s. The method was found to be applicable to routine analysis of both compounds in tablets. The coupling of the monolithic columns with a multi-syringe flow-injection analysis manifold provides an excellent and inexpensive tool to solve the separation problems without use of HPLC instrumentation.     

A multivariate approach for the simultaneous determination of losartan potassium and hydrochlorothiazide in a combined pharmaceutical tablet formulation

A convenient new method for the simultaneous determination of losartan potassium and hydrochlorothiazide, with minimum sample pretreatment, is described. The procedure, based on the multivariate analysis of spectral data in the 220−274 nm region by the partial least squares algorithm, is linear in the concentration range 1.06−5.70 mg L⁻¹ for hydrochlorothiazide and 4.0−22.2 mg L⁻¹ for losartan. It is simple, rapid and robust, allowing accurate and precise results, with drug recovery rates of 99.3 and 100.4% and relative standard deviations of 1.7 and 1.0% obtained for hydrochlorothiazide and losartan, respectively. The method was applied to the simultaneous determination of both analytes in tablets, and it provided good results which were in statistical agreement with those provided by independent HPLC analyses of the samples. The method has also been successfully applied for the construction of drug dissolution profiles of a commercial pharmaceutical preparation containing both analytes. Figure A UV-PLS method for the simultaneous determination of losartan potassium and hydrochlorothiazide in pharmaceutical tablet formulations has been developed and validated     

Chemometric tools in electroanalytical chemistry: Methods for optimization based on factorial design and response surface methodology

The aim of this paper is to give a brief overview of chemometric techniques based on factorial designs and response surface methodologies used in the optimization of electroanalytical methods. Chemometric techniques have several important advantages over one-way optimization for analytical applications, including a relatively low cost, a reduced number of experiments, and possibilities to evaluate interactions among variables. These techniques also enable the selection of optimal experimental conditions, helping to avoid trivial mistakes during optimization. Despite these facts, chemometric techniques have rarely been applied to electroanalytical data, especially in comparison with their use in spectroscopy. The application of chemometric methods in electroanalytical chemistry has been mostly used for solving overlapping signals, multivariate calibration methods, model identification and optimization of analytical procedures. This review is focused on the latter applications and overviews the role of full or fractional factorial designs (first-order designs), as well as second-order designs, such as central composite, Doehlert and Box-Behnken designs, for optimization of electroanalytical methods. A discussion of chemometric-related advantages is also given for stripping analyses, flow injection systems with amperometric detection, differential pulse voltammetry, square wave voltammetry and electrochemical sensor preparation.     

Multivariate optimization of a spectrophotometric method for copper determination in Brazilian sugar-cane spirits using the Doehlert design

This present work reports a multivariate optimization of a spectrophotometric method for copper determination in sugar-cane spirits, exploring the reaction of Cu(II) with phenylfluorone (PF) in the presence of cetylpyridinium chloride (CPC) and Triton X-100. The optimization strategy was based on the application of a two-level full factorial design for the screening of significant variables followed by a Doehlert design to determine the optimum conditions. The highest sensitivity was achieved when the pH was 8.0 and the PF and TX-100 concentrations were 6.2 × 10^− 5 and 1.0 × 10^− 2 mol L^− 1, respectively. The optimized method presented a limit of detection of 3.4 μg L^− 1 and a limit of quantification of 11 μg L^− 1 and was applied in the determination of Cu in 17 samples. The obtained results were compared with those obtained by ICP OES and no statistical difference between the methods was observed.     

Development and validation of a generic liquid chromatographic method for the simultaneous determination of five commonly used antimalarial drugs: Application to pharmaceutical formulations and human plasma

A simple, sensitive, and rapid liquid chromatographic method was developed and validated using diode array detection for the determination of five commonly used antimalarial drugs in pharmaceutical formulations and in human plasma. Chromatographic separation of antimalarial drugs and internal standard (ibuprofen) was achieved on a C₁₈ column with a mobile phase composed of 10 mM dipotassium orthophosphate at pH 3.0, methanol, and acetonitrile in a ratio of 20:38:42 v/v, at a flow rate of 1 mL/min. The analytes were monitored at 220 nm and separated in ?10 min. The method was validated for linearity, accuracy, precision, limit of quantification, and robustness. Both intra‐ and interday precisions (in terms of %RSD) were lower than 3% and accuracy ranged from 98.1 to 104.5%. Extraction recoveries were ≥96% in plasma. The limits of quantitation for artemether, lumefantrine, pyrimethamine, sulfadoxine, and mefloquine were 0.3, 0.03, 0.06, 0.15, and 0.15 μg/mL in human plasma. Stability under various conditions was also investigated. The method was successfully applied for quantification of antimalarial drugs in marketed formulations and in spiked human plasma. The method can be employed for routine QC purposes and in pharmacokinetic investigations.     

Development and validation of an LC method for the determination of emtricitabine and related compounds in the drug substance

A robust, precise, sensitive, linear, and simple RP LC method coupled with UV for the determination of emtricitabine or 2′,3′‐dideoxy‐5‐fluoro‐3′‐thiacytidine (FTC) and its related substances is described. The method uses an RP C18 column (25 cm×4.6 mm i.d.), 5 μm kept at a temperature of 35°C. The mobile phases for gradient elution consist of ACN, phosphate buffer (pH 4.4), and water. The flow rate is 1.0 mL/min and UV detection is performed at 280 nm. A system suitability test (SST) was developed to verify the adequate performance of the chromatographic system. The developed method was further validated with respect to robustness, precision, sensitivity, and linearity. A central composite design was applied to examine the robustness of the method. The method shows good precision, sensitivity, linearity, and robustness. Three commercial FTC samples were examined using this method. This method is suitable to be used for the determination of related substances and assay of FTC.     

Multivariate optimization of dispersive liquid–liquid microextraction for the determination of paclobutrazol and triflumizole in water by GC–MS

A new analytical method based on dispersive liquid–liquid microextraction with gas chromatography mass spectrometry has been optimized for the simultaneous determination of paclobutrazol and triflumizole in tap water and wastewater samples. A two‐level, full‐factorial design that allowed the study of main effects and factor interactions was applied to analyze the influence on microextraction process by chloroform, ethanol, potassium iodide and hand shaking period. The extraction conditions selected were 200 μL of chloroform, 3.0 mL of ethanol, 2.0 g of potassium iodide and 15 s of hand shaking. The limits of detection obtained for triflumizole and paclobutrazol under optimum conditions were 0.97 and 0.29 ng/mL, respectively. Calibration plots of both analytes were linear over a wide concentration range, and good precision was observed for replicate measurements. Applicability and accuracy of the method were determined by performing spiked recovery tests. Appreciable recovery results were obtained for municipal wastewater and matrix matching was used to obtain close to 100% recovery for tap water.     

Development and validation of an RP-HPLC method for quantitative determination of vanillin and related phenolic compounds in Vanilla planifolia

A simple and fast method was developed using RP-HPLC for separation and quantitative determination of vanillin and related phenolic compounds in ethanolic extract of pods of Vanilla planifolia. Ten phenolic compounds, namely 4-hydroxybenzyl alcohol, vanillyl alcohol, 3,4-dihydroxybenzaldehyde, 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin, p-coumaric acid, ferulic acid, and piperonal were quantitatively determined using ACN, methanol, and 0.2% acetic acid in water as a mobile phase with a gradient elution mode. The method showed good linearity, high precision, and good recovery of compounds of interest. The present method would be useful for analytical research and for routine analysis of vanilla extracts for their quality control.     

Simultaneous determination of losartan and active metabolite EXP3174 in rat plasma by HPLC with column switching

Summary A new liquid chromatographic method with columnswitching has been developed for the simultaneous determination of losartan and its active metabolite, EXP3174 in rat plasma. The plasma samaple was injected onto a precolumn of Lichroprep RP-8 after dilution with 5% acetonitrile in 50 mM phosphoric acid. Polar plasma components were eluted using this diluent. After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by an Inertsil ODS-2 column with acetonitrile-acetate buffer. The method showed excellent precision, accuracy and speed with detection limit 20 ng mL^–1. Total analysis time per sample was less than 40 min and the coefficients of variation for intra and inter-assay were 4.8%. This method has been successfully applied after oral administration of losartan to rat plasma samples.     

Multivariate technique for optimization of digestion procedure by focussed microwave system for determination of Mn, Zn and Fe in food samples using FAAS

This article describes the development by response surface methodology (RSM) of a procedure for iron, zinc and manganese determination by flame atomic absorption spectrometry (FAAS) in food samples after digestion employing a focussed microwave system. A Doehlert matrix was used to find optimal conditions for the procedure through response surface study. Three variables (irradiation power and time and composition of oxidant solution—HNO₃ + H₂O₂) were regarded as factors in the optimization study. The working conditions were established as a compromise between optimum values found for each analyte taking into consideration the robustness of the procedure. These values were 12 min, 260 W and 42% (v/v) for irradiation time, irradiation power and percent of H₂O₂ in solution, respectively. The accuracy of the optimized procedure was evaluated by analysis of certified reference materials and by comparison with a well-established closed vessel microwave dissolution methodology.     

Multiobjective optimization strategy based on desirability functions used for the microemulsion liquid chromatographic separation and quantification of norfloxacin and tinidazole in plasma and formulations

The aim of the present study was to optimize a microemulsion liquid chromatography method for the simultaneous determination of norfloxacin and tinidazole binary mixture using a chemometric protocol. Optimization experiments were conducted through a process of screening and optimization. A 2^7‐4 fractional factorial design was used as screening design. While the location of optimum conditions was established by applying Derringer's desirability function. The optimal mobile phase composition was predicted to be: 3.5% w/v SDS, 10.03% v/v 1‐propanol, 0.5% v/v 1‐octanol, and 0.3% triethylamine in 0.02 M phosphoric acid at pH 6.5. The mobile phase was delivered isocratically at a flow rate of 1 mL/min with UV detection at 290 nm. Tinidazole and norfloxacin were eluted with retention times of 1.8 and 5.8 min, respectively. The calibration plots displayed good linear relationships in the concentration ranges of 0.5–50 and 0.75–75 μg/mL for norfloxacin and tinidazole, respectively. The method was successfully applied for determination of both drugs in pharmaceutical dosage forms and real human plasma. Where the accuracy was proved by the low values of % error and high values of recovery, also the relative standard deviation for the results did not exceed 1.5%, proving the precision of the method.     

Microemulsion electrokinetic chromatography of drugs varying in charge and hydrophobicity: I. Impact of parameters on separation performance evaluated by multiple linear regression models

The separation of anionic, cationic and neutral drugs in microemulsion electrokinetic chromatography (MEEKC) was studied with a statistical experimental design. The concentration of sodium dodecyl sulfate (SDS, surfactant), 1-butanol (co-surfactant) and borate buffer and the factors Brij 35 (surfactant), 2-propanol (organic solvent) and cassette temperature were varied simultaneously, while the parameters pH (9.2), the concentration of octane (oil, 0.8% w/w), the voltage (10 kV) and the dimension of the fused-silica capillary, were kept constant. Eight different model substances were chosen with different hydrophobicities. Two of the analytes were positively charged, two were negatively charged, and the remaining four were neutral or close to neutral at the pH explored. The importance of each parameter on the separation window, the plate height and the retention factor for each of the analytes was studied by means of multiple linear regression (MLR) models. A new response was evaluated for anions, the quotient between the effective mobility in the microemulsion and the effective mobility in the corresponding buffer. Factors affecting selectivity changes were also explored, and it was found that SDS and 2-propanol had the largest effect on selectivity.     

Rapid determination of losartan and losartan acid in human plasma by multiplexed LC–MS/MS

A rapid LC–MS/MS method has been developed and validated for the determination of losartan (LOS) and its metabolite losartan acid (LA) (EXP‐3174) in human plasma using multiplexing technique (two HPLC units connected to one MS/MS). LOS and LA were extracted from human plasma by SPE technique using Oasis HLB® cartridge without evaporation and reconstitution steps. Hydroflumethiazide (HFTZ) was used as an internal standard (IS). The analytes were separated on Zorbax SB C‐18 column. The mass transition [M–H] ions used for detection were m/z 421.0 → 127.0 for LOS, m/z 435.0 → 157.0 for LA, and m/z 330.0 → 239.0 for HFTZ. The proposed method was validated over the concentration range of 2.5–2000 ng/mL for LOS and 5.0–3000 ng/mL for LA with correlation coefficient ?0.9993. The overall recoveries for LOS, LA, and IS were 96.53, 99.86, and 94.16%, respectively. Total MS run time was 2.0 min/sample. The validated method has been successfully used to analyze human plasma samples for applications in 100 mg fasted and fed pharmacokinetic studies.     

Development and validation of a HPLC method for the determination of voriconazole in pharmaceutical formulation using an experimental design

A rapid and sensitive RP-HPLC method with UV detection (260 nm) for routine analysis of voriconazole in a pharmaceutical formulation (Vfend^®) was developed. Chromatography was performed with mobile phase containing a mixture of acetonitrile and water (50:50, v/v) with flow rate was of 1.0 ml min⁻¹. Quantitation was accomplished with internal standard method. The procedure was validated for linearity (correlation coefficient = 0.9999), accuracy, robustness and intermediate precision. Experimental design was used for validation of robustness and intermediate precision. To test robustness, three factors were considered. Percentage of acetonitrile in mobile phase, flow rate and p^H; an increase in the flow rate results in a decrease of the drug found concentration, while the percentage of organic modifier and p^H have no important effect on the response. For intermediate precision measure the variables considered were: analyst, equipment and number of days. The R.S.D. value (0.45%, n = 24) indicated a good precision of the analytical method. The proposed method was simple, highly sensitive, precise and accurate and retention time less than 4 min indicating that the method is useful for routine quality control.     

Development and validation of an analytical method for detection of estrogens in water

An analytical procedure enabling routine analysis of four environmental estrogens at concentrations below 1 ng L^–1 in estuarine water samples has been developed and validated. The method includes extraction of water samples using solid-phase extraction discs and detection by gas chromatography (GC) with tandem mass spectrometry (MS–MS) in electron-impact (EI) mode. The targeted estrogens included 17- and 17-estradiol (aE2, bE2), estrone (E1), and 17-ethinylestradiol (EE2), all known environmental endocrine disruptors. Method performance characteristics, for example trueness, recovery, calibration, precision, accuracy, limit of quantification (LOQ), and the stability of the compounds are presented for each of the selected estrogens. Application of the procedure to water samples from the Scheldt estuary (Belgium – The Netherlands), a polluted estuary with reported incidences of environmental endocrine disruption, revealed that E1 was detected most frequently at concentrations up to 7 ng L^–1. aE2 was detected once only and concentrations of bE2 and EE2 were below the LOQ.Presented at the 9th FECS Conference on Chemistry and the Environment, Bordeaux, France, 29 August–1 September 2004     

Optimization of an analytical methodology for the determination of alkyl- and methoxy-phenolic compounds by HS-SPME in biomass smoke

A sampling and analysis method for the determination of 21 phenolic compounds in smoke samples from biomass combustion has been developed. The smoke is used to make smoked foods, following an artisanal procedure used in some parts of the Canary Islands. The sampling system consists of a Bravo H air sampler, two impingers, each one containing an aqueous solution of sodium hydroxide 0.1 mol L⁻¹, followed by a silica gel trap. The variables optimized to reach the best sampling conditions were volume of absorbent solution and sampling flow. Under the optimum conditions, 100 mL of absorbent solution of NaOH 0.10 mol L⁻¹ and 2 L min⁻¹ for the sampling flow, sampling efficiencies are higher than 80%. Analysis of phenolic compounds was carried out by headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography–mass spectrometry (GC-MS). Five different fiber coatings were employed in this study. By means of a central composite design, extraction time, salt concentration, and pH of the solution were optimized: 65-μm carbowax–divinylbenzene, extraction time 90 min, concentration in NaCl of 35% (m/v), and pH 2 yielded the highest response. Detection limits of phenol and their alkyl derivatives, guaiacol and eugenol, are between 1.13 and 4.60 ng mL⁻¹. 3-Methoxyphenol, 2,6-dimethoxyphenol, and vanillin have detection limits considerably higher. Good linearity (R ²≥0.98) was observed for all calibration curves in the established ranges. The reproducibility of the method (RSD, relative standard deviation) was found to oscillate between 7 and 18% (generally close or lower than 10%).     

Chemometrically assisted optimization and validation of a new HPLC method for the determination of paliperidone in pharmaceuticals

Paliperidone is an antipsychotic drug, which is used for the acute and maintenance treatment of schizophrenia. In this study, a new method was developed for the determination of Paliperidone in its extended-release tablets. Face-centered central composite design was applied for optimization of the method. Factors were decided as acetonitrile content, pH of the mobile phase and buffer concentration through preliminary studies. Optimal flow rate (1?mL/min), column temperature (35°C) and internal standard (Bupropion) were also determined during preliminary studies. Retention factors and tailing factors of Paliperidone and Bupropion were selected as responses. Derringer’s desirability function was applied for simultaneously optimization of these four responses. Optimal conditions were predicted as phosphate buffer (pH:3, 23?mM): acetonitrile (76:24, v:v). Developed method was validated in terms of linearity, detection and quantification limits, accuracy, precision, specificity and robustness. Method was found linear in the concentration range of 0.125-100?µg/mL. Mean equation of the calibration curve was y?=?0.0807 x - 0.0102 (R²?=?0.9999). Accuracy and precision of the method was evaluated with recovery values (98-102%) and relative standard deviation values (<2%), respectively. All other parameters were found acceptable. The method was successfully applied for the determination of Paliperidone in its extended-release tablets.     

Development and validation of a new analytical method for the determination of 1,4-dichlorobenzene in honey by gas chromatography-isotope dilution mass spectrometry after steam-distillation

A simple, fast, sensitive and robust analytical method using gas chromatography (GC)-isotope dilution mass spectrometry (MS) was developed and validated for the identification and quantification of 1,4-dichlorobenzene (p-DCB) residues in honey samples. The proposed methodology is based on steam-distillation using a Clevenger-type apparatus followed by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring (SIM) mode employing the isotopically labeled analogue d₄-1,4-dichlorobenzene (d₄-p-DCB) as internal standard (IS). Validation of the method was performed in two different GC-MS systems, using quadrupole MS (QMS) and ion-trap MS (ITMS) detectors, with no statistically significant differences between two. Recoveries were better than 91% with percent relative standard deviations lower than 12%. The instrumental limits of detection were 1 μg kg⁻¹ in the GC-ITMS system and 0.6 μg kg⁻¹ in the GC-QMS system. The expanded uncertainty was estimated as 17% at the currently accepted “action level” of 10 μg kg⁻¹. The method was applied to the analysis of 310 honey samples in an extensive national monitoring study. A quality control (QC) system applied during the assays has demonstrated a good performance and long-term stability over a period of more than 8 months of continuous operation.