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.     

Simultaneous Determination of Avermectin and Milbemycin Residues in Bovine Tissue by Pressurized Solvent Extraction and LC with Fluorescence Detection

A rapid and sensitive method has been developed for the simultaneous determination of four avermectins and one milbemycin residues in bovine tissue. The isolation of the analytes from muscle and liver samples was accomplished utilizing a pressurized solvent extractor. The optimized extraction procedure using acetonitrile/water (40:60, v/v) as extraction solvent, was automatically carried out at 100 °C and 10 MPa, applying two static cycles for 3 min. The extracts were cleaned up on a C₁₈ solid-phase extraction cartridge and analyzed by liquid chromatography with fluorescence detection after derivatization. Mean recoveries of the five analytes from fortified samples were between 84.8 and 101.8%, with relative standard deviations lower than 10.8%. The limit of detection and quantification were in the ranges of 0.1–0.2 and 0.5–0.6 μg kg^–1, respectively. The application of the newly developed method was demonstrated by analyzing bovine meat samples from market.     

Development and Validation of LC Coupled with SPE for Simultaneous Determination of Lamivudine, Oxymatrine and its Active Metabolite Matrine in Dog Plasma

A simple and highly selective method, based upon solid-phase extraction (SPE), ion-pair HPLC and UV absorbance detection, was developed and validated to determine lamivudine, oxymatrine and its active metabolite matrine in dog plasma. The analytes and famotidine (internal standard) were simultaneously extracted from plasma samples by SPE, and separated on a C18 column. The mobile phase consisted of acetonitrile-water (13:87, v/v, 5 mmol L^?1 sodium heptanesulfonate, at pH 3.2). The lower limit of quantification of the method was 0.1 mg L^?1 for these analytes. The linear calibration curves of the analytes were obtained in the concentration range of 0.1–40 mg L^?1. This method was successfully applied to the quantitative determination of plasma concentration of lamivudine, oxymatrine and its active metabolite matrine in dogs after single oral co-administration of 5.0 mg kg^?1 lamivudine and 30.0 mg kg^?1 oxymatrine.     

A environmentally friendly reversed-phase liquid chromatography method for phthalates determination in nail cosmetics

A simple and rapid analytical method was developed for the determination of phthalates, usually employed in nail cosmetic products. The method is based on an ultrasonic extraction of the sample with ethanol-water (90:10, v/v) followed by HPLC separation and quantitation. HPLC was carried out using a C18 column and spectrophotometric detection at 254 nm. A linear gradient elution was performed with ethanol-water starting from 50 to 95% ethanol in 30 min. Standard calibration curves were linear for all the analytes over the concentration range 5-200 μg ml⁻¹ with LOD values of about 0.5 μg ml⁻¹.The proposed green analytical method has been successfully applied for the analysis of commercial samples in order to check the presence of phthalates and to determine their concentration.     

Supercritical fluid extraction of triazine herbicides from solid matrices

Summary Several investigations were performed to optimise the extraction of polar triazine herbicides using supercritical fluid extraction from two different solid matrices: C₁₈-silica and soil samples. Supercritical CO₂ modified with methanol [10% (VV)] at 250 bars and 50°C was required to quantitatively extract Atrazine and 2-Hydroxyatrazine from spiked C₁₈-silica. Extraction of Desisopropyl-desethyl-2-hydroxyatrazine (MET) attained only 52%, even following addition of water to the polar modifier. Extractions of spiked soil samples (20 ppm of each pesticide) were successful at 300 bars and 65°C. A non polar wash improved the recoveries of the three target analytes [i.e. atrazine: 88%; 2-hydroxyatrazine: 96%; MET: 41%]. The extraction parameters employed are discussed in this paper.     

On-line solid-phase microextraction of triclosan, bisphenol A, chlorophenols, and selected pharmaceuticals in environmental water samples by high-performance liquid chromatography–ultraviolet detection

A method using on-line solid-phase microextraction (SPME) on a carbowax-templated fiber followed by liquid chromatography (LC) with ultraviolet (UV) detection was developed for the determination of triclosan in environmental water samples. Along with triclosan, other selected phenolic compounds, bisphenol A, and acidic pharmaceuticals were studied. Previous SPME/LC or stir-bar sorptive extraction/LC-UV for polar analytes showed lack of sensitivity. In this study, the calculated octanol–water distribution coefficient (log D) values of the target analytes at different pH values were used to estimate polarity of the analytes. The lack of sensitivity observed in earlier studies is identified as a lack of desorption by strong polar–polar interactions between analyte and solid-phase. Calculated log D values were useful to understand or predict the interaction between analyte and solid phase. Under the optimized conditions, the method detection limit of selected analytes by using on-line SPME-LC-UV method ranged from 5 to 33 ng?L^?1, except for very polar 3-chlorophenol and 2,4-dichlorophenol which was obscured in wastewater samples by an interfering substance. This level of detection represented a remarkable improvement over the conventional existing methods. The on-line SPME-LC-UV method, which did not require derivatization of analytes, was applied to the determination of TCS including phenolic compounds and acidic pharmaceuticals in tap water and river water and municipal wastewater samples.

Extraction of neurotransmitters from rat brain using graphene as a solid-phase sorbent, and their fluorescent detection by HPLC

A simple, cost-effective and efficient method was developed for the determination of glycine, gamma-aminobutyrate and taurine in rat brain using graphene as a sorbent for solid-phase extraction. The analytes were eluted from a graphene-packed solid-phase extraction cartridge with methanol, derivatized at their amino groups with the fluorescent label 4-carboxy-2,6-dimethylquinoline N-hydroxysuccinimide ester, and then separated and fluorescently detected by HPLC. The type and volume of eluent, sample pH, extraction time and sample volume were optimized with respect to sensitivity and precision. Under optimal conditions, linear response is obtained in the concentration range from 0.1 to 50?μg?g?1, with correlation coefficients of >0.990. The limits of detection are 23.4?ng?g^?1 (gamma-aminobutyrate), 45.3?ng?g^?1 (glycine) and 67.5?ng?g^?1 (taurine) (S/N?=?3). The results reveal the potential of graphene as a sorbent in the analysis of biological samples.

Analysis of zearalenone and α-zearalenol in animal feed using high-performance liquid chromatography

Different extraction and clean-up techniques used before HPLC analysis were compared in order to obtain a reliable method for the quantitative determination of zearalenone (ZEA) and α-zearalenol (α-ZOL) in animal feed. Immunoaffinity clean-up was compared to C₁₈ and Florisil column clean-up. Extracted samples were analysed by reversed-phase HPLC with fluorescence detection (λ_ex=274 nm, λ_em=440 nm). A mobile phase of acetonitrile:water (50:50 (v/v)) and a flow-rate of 1.0 ml min⁻¹ resulted in a good separation between ZEA and α-ZOL. Using immunoaffinity clean-up the linear range was between 25 and 600 μg kg⁻¹ for ZEA and α-ZOL in maize. Intra-laboratory coefficients of variation (CV) (under repeatability conditions) were 9.16% for ZEA and 2.18% for α-ZOL. Recoveries for spiked ZEA and α-ZOL samples ranged from 89 to 110% with CVs between 5.2 and 11.2% (under within-laboratory reproducibility conditions). Using C₁₈ and Florisil solid-phase clean-up, matrix interference was too high. Therefore, naturally contaminated animal feed samples were analysed using the developed HPLC method coupled to the immunoaffinity clean-up.     

Sensitive determination of carbamates in fruit and vegetables by a combination of solid-phase extraction and dispersive liquid-liquid microextraction prior to HPLC

We report on a new method for sample pretreatment. It is based on solid-phase extraction combined with dispersive liquid-liquid microextraction (SPE-DLLME) and was applied to the determination of carbamates in apple and cucumber. The carbamates carbofuran, carbaryl, and pirimicarb were first extracted from samples by SPE. The analytes were then eluted from the sorbent with acetonitrile, and the DLLME technique was then applied to the resulting eluate. Finally, the analytes in the extraction solvent were determined by HPLC. Several parameters affecting the SPE-DLLME process were optimized. The new method provides enrichment factors that range from 5,400 to 7,650. Calibration plots are linear in the range from 0.25 to 100?μg?kg^?1 for carbofuran and carbaryl, and from 0.10 to 100?μg?kg^?1 for pirimicarb, with correlation coefficient (r²) ranging from 0.9980 to 0.9997. The limits of detection range from 5 to 60?pg?kg^?1 (at S/N?=?3). The method was successfully applied to the extraction and sensitive determination of carbamates in apple and cucumber samples.

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%).     

Determination of aromatic amines in environmental water samples using solid-phase extraction modified with sodium dodecyl sulphate and micellar liquid chromatography

Extraction and determination of seven aromatic amines in environmental water samples were performed with solid-phase extraction (SPE) and micellar liquid chromatography (MLC) using experimental design. Extraction of aromatic amines was carried out with a C₁₈ cartridge modified with sodium dodecyl sulphate (SDS). The washing solution and elution solvent for extraction of aromatic amines were aqueous solution containing 5% (v/v) acetonitrile and 5% (v/v) acetone and 3 mL methanol, respectively. The chemometrics approach was applied for the separation optimisation of these compounds using MLC. Different mobile phase compositions were used for modelling based on retention times to obtain the best separation using central composite design. The optimum mobile phase composition for separation and determination of analytes in water samples was 69 mM SDS, 9% v/v 1-propanol and pH = 6.4. Recoveries were between 84.8–93.5% with relative standard deviation (RSD) less than 5.8% (n = 5). Limits of detection and linear range were 1–4.5 and 3.1–125.0 µg/L, respectively. The proposed method was applied to determine the aromatic amines in real samples (river and well waters). Amount of 4-nitroaniline and 3-nitroaniline in river water sample were 2.15 and 1.91 µg/L, respectively.     

Analysis of methylamine by solid-phase microextraction and HPLC after on-fibre derivatization with 9-fluorenylmethyl chloroformate

A method for the determination of methylamine (MA) in aqueous matrices is reported which uses solid-phase microextraction (SPME) for enrichment and derivatization of the analyte, and high performance liquid chromatography (HPLC). The fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC) has been used for derivatization. The SPME fibres were successively immersed in the samples and in the derivatization solutions to extract MA and FMOC, respectively. After a defined time of reaction, the derivatized analyte was desorbed into the chromatographic system, and chromatographed in a LiChrosphere 100 RP18, i.d., 5 μm, column under gradient elution. In order to improve the MA-FMOC peak profile, a precolumn ( i.d., packed with Hypersil C₁₈ phase, 30 μm) was connected on-line to the analytical column by means of a switching valve. The experimental conditions (including fibre coating, times of adsorption, reaction and desorption, and concentration of reagent) have been optimised, and the results have been compared with those achieved by using a method previously validated for aliphatic amines in which extraction and derivatization were carried into C₁₈ solid-phase extraction (SPE) cartridges. Although less sensitive, the SPME based method allowed the quantification of MA over the range 2.5-10.0 μg/ml with linearity, reproducibility and accuracy comparable to that of the SPE based method, the limit of detection being 0.75 μg/ml. The main advantages of the proposed SPME procedure are: sample handling involved in the extraction and derivatization steps was considerably reduced, it was free organic solvent and non-destructive. Moreover, the proposed conditions allowed the selective determination of MA in the presence of other primary and secondary short-chain aliphatic amines. The utility of the proposed procedure for the quantification of MA in different types of waters is discussed.     

LC-MS/MS method for the simultaneous quantification of artesunate and its metabolites dihydroartemisinin and dihydroartemisinin glucuronide in human plasma

Artesunate (AS), a hemisuccinate derivative of artemisinin, is readily soluble in water and can easily be used in formulations for parenteral treatment of severe malaria. AS is rapidly hydrolyzed to the active metabolite dihydroartemisinin (DHA) and primarily eliminated by biliary excretion after glucuronidation. To investigate systematically the AS metabolism and pharmacokinetics, a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of AS and its metabolites DHA and DHA glucuronide (DHAG) in human plasma samples was developed. Compared to previous methods, our method includes for the first time the quantification of the glucuronide metabolite using a newly synthesized stable isotope-labeled analogue as internal standard. Sample preparation was performed with only 50 μL plasma by high-throughput solid-phase extraction in the 96-well plate format. Separation of the analytes was achieved on a Poroshell 120 EC-C₁₈ column (50*2.1 mm, 2.7 μm, Agilent Technologies, Waldbronn, Germany). The method was validated according to FDA guidelines. Calibration curves were linear over the entire range from 1 to 2,500 nM (0.4–961.1 ng/mL), 165 to 16,500 nM (46.9–4,691.8 ng/mL), and 4 to 10,000 nM (1.8–4,604.7 ng/mL) for AS, DHA, and DHAG, respectively. Intra- and interbatch accuracy, determined as a deviation between nominal and measured values, ranged from ?5.7 to 3.5 % and from 2.7 to 5.8 %, respectively. The assay variability ranged from 1.5 to 10.9 % for intra- and interbatch approaches. All analytes showed extraction recoveries above 85 %. The method was successfully applied to plasma samples from patients under AS treatment.

Determination of Visnagin inAmmi visnagaHairy Root Cultures Using Solid-Phase Extraction and High-Performance Liquid Chromatography

A high-performance liquid chromatographic method was developed for separation of the furochromone fraction and for determination of visnagin inAmmi visnagahairy root cultures. Lyophilized samples were extracted with chloroform:methanol (1:1, v/v) and purified on solid-phase extraction cartridges. HPLC analyses were performed on a Eurospher 100-C₈Knauer column and the mobile phase was 29:28:526:417 (v/v/v/v) acetonitrile:tetrahydrofuran:30 mM citric acid (pH 3.0):methanol. Quercetin was used as internal standard. Peaks were identified by addition of authentic standards and/or by diode-array detection.     

Determination of the immunosuppressive drug tacrolimus in its dosage forms by high-performance liquid chromatography

Summary A high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of FK506, a new immunosuppressant, in bulk drug samples and dosage forms. Equilibration between FK506 (I) and its tautomeric compounds (II and III) was accomplished artificially in water-dehydrated alcohol (11) employed as an extraction solvent. After reaching equilibrium, separation of I, treated as the representetive of equilibrated FK506, from its related substances was achieved by reversed-phase HPLC on a C₁₈ column with water-isopropyl alcohol-tetrahydrofuran (522, v/v) as the mobile phase, and detection at 220 nm. Component I in dosage forms could be recovered satisfactorily and determined with good precision. The calibration graph was linear over the range 2–6 g for I.     

HPLC determination of perfluorinated carboxylic acids with fluorescence detection

Perfluorinated carboxylic acids (PFCAs) represent an important group of persistent perfluorinated organic compounds commonly determined in environmental and biological samples. A reversed-phase HPLC method was developed based on derivatization of the PFCAs with the commercially available fluorescent reagent 3-bromoacetyl coumarin. The method was optimized and this resulted in the efficient separation of PFCAs containing from 3 to 12 carbon atoms in molecule in 25 min run. To improve sensitivity, the preconcentration step has been optimized using Oasis-WAX and C18 sorbents for SPE. A 100-fold preconcentration is achieved by solid-phase extraction with the sorbent C18 Sep-PAK to result in limits of detection in the range from 43 to 75 ppt for the analytes examined, and in the application of the method of water analysis.

Determination of fifteen priority phenolic compounds in environmental samples from Andalusia (Spain) by liquid chromatography–mass spectrometry

This work describes the optimisation of a method to determinate fifteen phenolic compounds in waters, sediments and biota (green marine algae) by liquid chromatography coupled to mass spectrometry (LC-MS) with atmospheric pressure chemical ionisation (APCI) in the negative mode. The LC separations of the studied compounds and their MS parameters were optimised in order to improve selectivity and sensitivity. Separation was carried out with a C₁₈ column using methanol and 0.005% acid acetic as mobile phase in gradient mode. The molecular ion was selected for the quantitation in selective ion monitoring (SIM) mode. A solid-phase extraction (SPE) method was applied in order to preconcentrate the target analytes from water samples. However, extraction of the compounds from sediment and biota samples was carried out by liquid–solid extraction with methanol/water after studying the influence of other organic solvents. In addition, a clean-up step by SPE with HLB Oasis cartridges was necessary for sediments and biota. The proposed analytical methodology was validated in the target environmental matrices by the analysis of spiked blank matrix samples. Detection limits were 10–50 ng L^–1 for water, 1–5 g kg^–1 for sediments and 2.5–5 g kg^–1 for biota samples. Good recoveries and precision values were obtained for all matrices. This methodology has been successfully applied to the analysis of incurred water, sediment and biota samples from Andalusia (Spain).     

Determination of Carotenoids in the Chinese Medical Herb Jiao-Gu-Lan (Gynostemma Pentaphyllum MAKINO) by Liquid Chromatography

A QSTAR Pulsar quadrupole time-of-flight mass spectrometer was used for the determination of chlorophenols in surface water samples. The investigated compounds were: 2-chlorophenol; 4-chloro-3-methylphenol; 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol. Each analyte is listed by the US-EPA as a priority pollutant. Sample enrichment of water samples was achieved by a solid-phase extraction procedure, using a Waters Oasis HLB cartridges followed by LC-Tandem-MS. A narrow-bore 2.1-mm-i.d. reversed phase LC C-18 column operating with a mobile phase flow rate of 0.2 mL min^–1. was used to separate the analytes. The whole column effluent was diverted to the ion spray interface source. For the determination of the analytes the hybrid quadrupole time-of-flight spectrometer operated in product ion scan acquisition mode. Average recoveries from 2 L samples varied from 91 to 110% and relative standard deviations (RSD) were less than 10% for all samples. The limit of detection (signal-to-noise ratio=3) of the method for the phenols in drinking water samples is less than 10 ng L^–1. In real environmental samples, levels of the selected analytes varied from non-detected up to 0.5 g L^–1 for pentachlorophenol.     

HPLC Determination of the Antiseptic Agent Chlorhexidine and its Degradation Products 4-Chloroaniline and 1-Chloro-4-Nitrobenzene in Serum and Urine

Chlorhexidine (1,1-hexamethylene-bis-[5-(p-chlorophenyl)-biguanide]-digluconate; CHD) is one of the most frequently used antiseptics. In spite of this, its resorption and elimination has hardly been investigated. One of the main reasons for this is the lack of suitable procedures for its analysis in biological materials. Samples of CHD also may contain the carcinogen 4-chloroaniline which also may be formed as a degradation product in preparations.A HPLC method with end-capped stationary phase was developed which separates CHD, 4-chloroaniline and 1-chloro-4-nitrobenzene (a degradation product of 4-chloroaniline). Methods of solid-phase extraction and deproteinization for the purpose of isolating the analytes in serum and urine were also tested. In the latter process, isolation of all three compounds from the biological material using a single procedure was unsuccessful. Isolation of CHD, on the other hand, both from serum and from urine using solid-phase extraction with weak cation exchangers was successful and showed reliable retrieval rates.     

Toxicological screening of human plasma by on‐line SPE‐HPLC‐DAD: identification and quantification of acidic and neutral drugs

A multi‐analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on‐line solid‐phase extraction (SPE)–HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro‐neutral, one strong anion‐exchange, one weak cation‐exchange) for on‐line extraction, five HPLC‐columns [one C₁₈ (GeminiNX), two phenyl‐hexyl (Gemini C₆‐Phenyl, Kinetex Phenyl‐Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre‐treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion‐exchanger SPE cartridge (StrataX‐A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C₆‐Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile–water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter‐/intra‐day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from ?14 to 10%. A computer‐assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi‐drug intoxications are presented. Copyright © 2015 John Wiley & Sons, Ltd.