Optimisation of a CE method for caffeine analysis in decaffeinated coffee

Multiple response simultaneous optimisation was used to develop a micellar electrokinetic chromatography method for caffeine determination in decaffeinated coffees. Buffer composition and voltage were optimised using a central composite design. Six responses were evaluated and each set of response values was regressed on the factor levels of the experimental design using linear and quadratic models. The regression models, correlation coefficients and a principal component analysis (PCA) were used to determine the optimum conditions. Successful results were obtained using a buffer containing 10 mmol L⁻¹ sodium carbonate and 50 mmol L⁻¹ sodium dodecylsulfate, 15 kV voltage, 25 °C temperature, 48 cm × 50 μm fused-silica capillary, hydrodynamic injection of 50 mBar during 7 s and detection at 206 nm. This optimised method was applied for caffeine analysis in 45 samples of commercial decaffeinated coffee.     

Multiresidue method for analysis of pesticides in pepper and tomato by gas chromatography with nitrogen–phosphorus detection

An analytical multiresidue method for the simultaneous determination of various classes of pesticides in vegetables (pepper and tomato) was developed. Vegetable samples are extracted with acetone and the pesticides are partitioned into ethyl acetate/cyclohexane. Final determination was made by gas chromatography with nitrogen–phosphorus detection. Confirmation analysis of pesticides was carried out by gas chromatography coupled with mass spectrometry in the selected ion monitoring (SIM) mode. The identification of compounds was based on retention time and on comparison of the primary and secondary ions. Recovery studies were performed at 0.05, 0.1 and 0.02 mg kg⁻¹ fortification levels of each compound and the recoveries obtained ranged from 70.1% to 128.5% with relative standard deviations lower than 7%. The method showed good linearity over the range assayed 50–1500 μg l⁻¹ and the detection and quantification limits for the pesticides studied varied from 0.1 to 4.4 μg kg⁻¹ and 0.4 to 14.5 μg kg⁻¹, respectively. The proposed method was used to determine pesticides levels in peppers and tomatoes grown in experimental greenhouses.     

Analyses of selected non-authorized insecticides in peppers by gas chromatography/mass spectrometry and gas chromatography/tandem mass spectrometry

Two methods based on gas chromatography coupled with mass spectrometry and tandem mass spectrometry analyzers are described for the identification, confirmation and quantitation of two EU-banned insecticides: isocarbophos and isofenphos-methyl, detected in recent monitoring programmes in pepper samples. The proposed methodologies involved a liquid–liquid extraction with acetonitrile followed by a cleanup step by dispersive solid-phase extraction using primary–secondary amine as sorbent material. Recovery studies performed on peppers spiked at different fortification levels (10 and 50 μg kg⁻¹) yielded average recoveries in the range 85–98% with RSD values below 8%. Identification, confirmation and quantitation were carried out by gas chromatography/mass spectrometry (GC–MS) in selected ion monitoring mode and gas chromatography/tandem mass spectrometry (GC–MS/MS) using an ion trap operating in the multiple reaction monitoring (MRM) mode. The obtained limits of detection (LODs) were in the range 0.1–0.3 μg kg⁻¹, depending on the technique. The proposed methods were successfully applied to the analysis of suspected pepper samples.     

Quantitative analysis of lincomycin and narasin in poultry, milk and eggs by liquid chromatography-tandem mass spectrometry

This study presents the simultaneous extraction and determination of lincomycin (LCM) and narasin (NAR) by using liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-MS/MS) on samples from poultry, milk and eggs (n = 196). The homogenised samples are extracted with acetonitrile and the extract is further cleaned using C₁₈ solid-phase extraction cartridges. The recoveries of the analytes in different matrices were found ranging from 90% to 101% and 85% to 95% for LCM and NAR, respectively. The corresponding limits of detection were 0.6 and 1.5 ng g⁻¹ for LCM and NAR, respectively. As a result of monitoring, NAR was not detected in any samples and LCM was detected in one egg with a concentration of 25 ng g⁻¹. The method was relatively simple to perform and therefore could be used for food safety surveillance activities.     

Simplified pesticide multiresidues analysis in fish by low-temperature cleanup and solid-phase extraction coupled with gas chromatography/mass spectrometry

A simple and fast method for the simultaneous analysis of thiobencarb, deltamethrin and 19 organochlorine pesticide residues in fish by gas chromatography–mass spectrometry was investigated in this study. Samples are extracted with acetonitrile. Most of lipids in the extract are eliminated by low-temperature cleanup, prior to solid-phase extraction cleanup. The lipids extracted from the fish samples were easily removed without any significant losses of the pesticides. Aminopropyl (NH₂) cartridge was effective to eliminate the remaining interference. Spiked experiments were carried out to determine the recovery, precision and limits of detection (LODs) of the method. The method detection limits ranged from 0.5 μg kg⁻¹ to 20 μg kg⁻¹, whilst recoveries of the pesticides were in the range of 81.3–113.7% with relative standard deviations ?13.5% at a spiked concentration of 0.05 mg kg⁻¹, 0.02 mg kg⁻¹ and 0.1 mg kg⁻¹. The newly developed method is demonstrated to give efficient recoveries and LODs for detecting pesticide multiresidues in fish.     

A validated matrix solid-phase dispersion method for the extraction of organophosphorus pesticides from bovine samples

A method based on matrix solid-phase dispersion (MSPD) was developed for the quantitative extraction of five organophosphorus (OPPs) pesticides from bovine samples. The determination was carried out by high performance liquid chromatography (HPLC) with diode array spectrophotometric UV detection. The MSPD extraction with octadecylsilyl (C18) sorbent combined with a silica gel clean-up and acetonitrile elution was optimised for chlorpyrifos, chlorfenvinphos, diazinon, fenitrothion, and parathion-methyl. The method was validated, yielding recovery values higher than 94%, except for chlorfenvinphos in liver (55%), and precision values, expressed as relative standard deviations (RSDs), which were less than or equal to 15% in liver and 11.5% in muscle at spiking levels of 0.25, 2.5 and 5 μg g⁻¹. Linearity was studied from 0.5 to 15 μg g⁻¹, and the limits of detection (LODs) were found to be lower than 0.1 μg g⁻¹_. This method was applied to the analysis of real samples with confirmative analyses performed using gas chromatography–mass spectrometry (GC–MS) in selected ion monitoring mode (SIM).     

Simultaneous determination of diethylene glycol,diethylene glycol monoethyl ether,coumarin and caffeine in food items by gas chromatography

A simple gas chromatography–flame ionisation method was initially developed for the simultaneous determination of the prohibited flavours, namely diethylene glycol (DEG), diethylene glycol monoethyl ether (DEGME) and coumarin in food samples. The analytes were extracted using methanol, and was sonicated for 10 min. The extracts were filtered and directly injected into the GC unit that was fitted with a capillary AT^TM-AQUAWAX column. Caffeine, which was found in some of the soft drink samples were also successfully separated. Excellent separation of these flavours and caffeine was achieved in about 23 min. Limit of detection, linear range, and reproducibility of the retention time were evaluated. Average recoveries in the ranges of 93.44–97.54% (RSD, 2.68%) for DEGME, 92.99–101.45% (RSD, 2.99%) for DEG, 90.64–100.00% (RSD, 1.99%) for coumarin and 94.62–97.50% (RSD, 2.54%) for caffeine were obtained. None of the food items analysed was found to contain coumarin, DEG or DEGME. However, of the 35 soft drinks and fruit juices that were analysed, eleven samples were found to contain caffeine, but only one exceeded the legal limit.     

Determination of theobromine,theophylline and caffeine in cocoa samples by a high-performance liquid chromatographic method with on-line sample cleanup in a switching-column system

A simple reversed-phase high-performance liquid chromatographic method was developed for the determination of theobromine, theophylline and caffeine in cocoa samples. In the sample cleanup step, the procedure involves an on-line solid-phase extraction of analytes from cocoa samples into a home-made dry-packed pre-column with ODS-C₁₈ using a column-switching system. The separation was performed on a C₁₈ Nova-Pak column (150 mm × 3.9 mm, 4 μm) using a mobile phase consisting of a solution of 20% of methanol in water under isocratic conditions, at a flow-rate of 1.4 ml/min. The validation method revealed quantitative recoveries (>95.0%) with a coefficients of variation <3.2% and it also provided a good precision for data validation. The overlap of sample cleanup, analysis and recondition of the precolumn increases the sample throughput to 8 samples/h. Furthermore, the proposed method was successfully applied to analysis of cocoa samples “Trinitario”, “Forastero” and “Criollo” grown in different seasons of the year and fermented for 3 and 7 days, respectively. The results showed a slight reduction in the theobromine and caffeine content according to the fermentation times. In the same way, the theobromine/caffeine ratio was assessed, with the purpose of establishing a correlation with the genotype of the studied samples.     

FT-NIR spectroscopy for caffeine estimation in instant green tea powder and granules

The feasibility of measuring caffeine content in instant green tea and granules was investigated by Fourier Transform Near-Infrared (FT-NIR) spectroscopic technique. A calibration model was developed using pure caffeine standards of varying concentrations in the near-infrared region (4000-12000 cm⁻¹). The developed model was validated using test validation technique. FT-NIR spectroscopy with chemometrics, using the PLS-first derivative plus straight line subtraction method could predict the caffeine content in tea samples accurately up to an R² value greater than 0.98 and a standard error of prediction (SEP) value less than 2.0 with 6 factors in the prediction model. The developed model was applied to predict caffeine content in tea samples within 2-5 min. The developed procedure was further validated by recovery studies by comparing with UV spectroscopic method of caffeine determination.     

Determination of Histamine and Tyramine in Canned Fish Samples by Headspace Solid-Phase Microextraction Based on a Nanostructured Polypyrrole Fiber Followed by Ion Mobility Spectrometry

This paper describes the development of a simple, rapid, and selective method based on headspace solid-phase microextraction (HS-SPME) combined with ion mobility spectrometry (IMS) for the simultaneous determination of histamine (HIS) and tyramine (TYR) in the canned fish samples. The spectra interferences were eliminated by using a new alternate reagent gas and resulted in an increased sensitivity and selectivity of IMS technique. A dodecylbenzenesulfonate-doped polypyrrole coating was used as a fiber for HS-SPME method. The calibration curves were linear in the range of 30–300 ng g^?1 (R ² > 0.994). Limits of detection for HIS and TYR were 3 and 4 ng g^?1, respectively. The proposed method was successfully applied to determine HIS and TYR in different canned fish samples without derivatization steps. Method validation was conducted by comparing our results with those obtained through gas chromatography-mass spectrometry method.     

Quantification of (+)-catechin and (−)-epicatechin in coconut water by LC–MS

An analytical technique has been developed to detect trace amounts of both (+)-catechin and (−)-epicatechin in the coconut water extract. Both (+)-catechin and (−)-epicatechin in the coconut water were found for the first time by the solid-phase extraction, and they were further analysed using liquid chromatography (LC)–ion trap mass spectrometry (MS) equipped with positive atmospheric pressure chemical ionisation interface on multiple reaction monitoring mode. The limit of detection and quantification for (+)-catechin were 7.8 and 15.6 μg/ml, respectively, while those for (−)-epicatechin were 3.9 and 7.8 μg/ml, respectively. The average concentration of (+)-catechin and (−)-epicatechin in the coconut water was 0.344 and 0.242 μg/ml, respectively. The LC–MS/MS analysis accelerated the quantitative analysis of (+)-catechin and (−)-epicatechin in the coconut water extract with high accuracy, precision and recovery. Results obtained in this study will serve as quality control and useful reference for drug development.     

Double-antibody based immunoassay for the detection of β-casein in bovine milk samples

The concentration of casein (CN) is one of the most important parameters for measuring the quality of bovine milk. Traditional approach to CN concentration determination is Kjeldahl, which is an indirect method for determination of total nitrogen content. Here, we described a double-antibody based direct immunoassay for the detection of β-CN in bovine milk samples. Monoclonal antibody (McAb) was used as capture antibody and polyclonal antibody (PcAb) labelled with horseradish peroxidase (HRP) as detection antibody. With the direct immunoassay format, the linear range of the detection was 0.1–10.0 μg mL⁻¹. The detection limit was 0.04 μg mL⁻¹. In addition, the concentration of β-CN in real bovine milk samples has been detected by the developed immunoassay. There was a good correlation between the results obtained by the developed technique and Kjeldahl method from commercial samples. Compared to the traditional approach, the advantage of the assay is no need of time-consuming sample pretreatment.     

Simultaneous determination of aspartame,cyclamate, saccharin and acesulfame-K in soft drinks and tabletop sweetener formulations by capillary electrophoresis with capacitively coupled contactless conductivity detection

Capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) was used for simple, rapid, and simultaneous determination of aspartame, cyclamate, saccharin and acesulfame-K in commercial samples of soft drinks and tabletop sweetener formulations. A buffer solution containing 100 mmol L⁻¹ tris(hydroxymethyl)aminomethane (TRIS) and 10 mmol L⁻¹ histidine (His) was used as background electrolyte (BGE). A complete separation of the analytes could be attained in less than 6 min. The limits of detection (LOD) and quantification (LOQ) were considered better than those usually obtained by CE with photometric detection. Recoveries ranging from 94% to 108% were obtained for samples spiked with standard solutions of the sweeteners. The relative standard deviation (RSD) for the analysis of the samples with the CE-C⁴D method varied in the range of 1.5%–6.5%.     

Analysis of volatile nitrosamines from a model system using SPME–DED at different temperatures and times of extraction

Solid-phase microextraction (SPME) coupled to a direct extraction device (DED) was evaluated as a method for extracting nitrosamine (NA) standard mix containing nine volatile nitrosamines from a solid food model system using several temperatures and times of extraction. The efficacy of extraction, the linearity of response and the sensitivity of this method for analysis of NA were determined at refrigeration (4 °C) and room (25 °C) temperature. Several extraction times (15, 30, 60,120 and 180 min) were also tested. Analysis was carried out with gas chromatography–mass spectrometry (GC–MS) in selected ion monitoring (SIM) mode. At 4 °C all NA were detected at all concentrations studied (ranged from 1 to 50 ng ml⁻¹) except for N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) and N-nitrosodiphenilamine (NDPheA). Better results were obtained at 25 °C in terms of efficacy of extraction, linearity values (regression coefficients, R²) and sensitivity values (limit of detection, LOD). Only 15 min of extraction time were enough to extract all NA from gelatines (10 ng ml⁻¹) at 25 °C. None of the NA reached the equilibrium, even when long times of extraction (20 and 24 h) were evaluated. SPME–DED appears to be a rapid and suitable technique for extracting NA from model food system at both refrigeration and room temperatures.     

Analysis of pesticide residues in seaweeds using matrix solid-phase dispersion and gas chromatography–mass spectrometry detection

Products containing organophosphates, carbamates and pyrethroids pesticides, employed as chemotherapeutants in aquaculture, can remain as residues in the marine environment. Matrix solid-phase dispersion (MSPD) was developed to extract seventeen pesticides from seaweed samples using Florisil and graphitized carbon black as clean-up adsorbents prior to gas chromatography–mass spectrometry (GC–MS) determination. The extraction has been optimised by a Box–Behnken design. The optimal conditions were 1 g of seaweed sample, 4 g of anhydrous sodium sulphate as dispersant, 3.6 g of Florisil and 0.4 g of GCB and an elution volume of 14 mL of a hexane:ethyl acetate mixture containing 40% ethyl acetate. The recoveries ranged from 81.6% to 113.2% with relative standard deviations (RSDs) ranging from 1.6% to 13.2%. The limits of detection (LOD) ranged from 0.5 to 2.9 ngg⁻¹. Internal quality control was successfully carried out to verify the quality of the data obtained in the analysis of these pesticides in seaweed samples.     

Analysis of some selected catechins and caffeine in green tea by high performance liquid chromatography

Green tea seems to have a positive impact on health due to the catechins-found as flavanols. Thus, the present study was aimed to develop a low cost reversed phase high performance liquid chromatographic (HPLC) method for simultaneous determination of flavanol contents, namely catechin (C), epicatechin (EC), epigallocatechin (EGC), epicatechin 3-gallate (ECG) and epigallocatechin 3-gallate (EGCG) and caffeine in 29 commercial green tea samples available in a Saudi Arabian local market. A C-18 reversed-phase column, acetonitrile–trifluoroacetic acid as a mobile phase, coupled with UV detector at 205 nm, was successfully used for precise analysis of the tested analytes in boiled water of digested tea leaves. The average values of N (No. of theoretical plates), HETP (height equivalent of theoretical plates) and R_s (separation factor) (at 10 μg ml⁻¹ of the catechins EC, EGC, EGCG and ECG) were 2.6 × 10³ ± 1.2 × 10³, 1.7 × 10⁻³ ± 4.7 × 10⁻⁴ cm and 1.7 ± 5.53 × 10⁻², respectively. The developed HPLC method demonstrated excellent performance, with low limits of detection (LOD) and quantification (LOQ) of the tested catechins of 0.004–0.05 μg ml⁻¹ and 0.01–0.17 μg ml⁻¹, respectively, and recovery percentages of 96–101%. The influence of infusion time (5–30 min) and temperature on the content of the flavanols was investigated by HPLC. After a 5 min infusion of the tea leaves, the average concentrations of caffeine, catechin, EC, EGC, ECG and EGCG were found to be in the ranges 0.086–2.23, 0.113–2.94, 0.58–10.22, 0.19–24.9, 0.22–13.9 and 1.01–43.3 mg g⁻¹, respectively. The contents of caffeine and catechins followed the sequence: EGCG > EGC > ECG > EC > C > caffeine. The method was applied satisfactorily for the analysis of (+)-catechin, even at trace and ultra trace concentrations of catechins. The method was rapid, accurate, reproducible and ideal for routine analysis.     

Green tea: A promising anticancer agent for renal cell carcinoma

Renal cell carcinoma (RCC) is one of the most lethal amongst the urologic malignancies, comprising three percent of all human neoplasms, and its incidence appears to be rising. RCC is refractory to both chemotherapy and radiotherapy. Therefore, the discovery of new strategies for therapeutic intervention remains a priority. Green tea (Camellia sinensis) and tea polyphenols have been proposed to exert protective effects against several types of cancer, based on preclinical and clinical trial data; however, the anticarcinogenic activity of green tea towards RCC is unknown. In this study, a targeted metabolite analysis on a green tea leaves methanolic extract was performed by HPLC/DAD and the antiproliferative activity of the extract was assayed using human renal cancer cell lines A-498 and 769-P. The total phenolic content was very high (31.8% of methanolic extract), and the main compounds were flavan-3-ols (94.3% of the total phenolic content), and especially (−)-epigallocatechin-3-gallate (35.9% of the total phenolic content). In addition, two methylxanthines – theophylline and caffeine – were also present in the extract, caffeine being the most abundant. Green tea extract strongly inhibited the growth of both RCC cell lines in a concentration-dependent manner, with IC₅₀ values of 54 ± 10 and 129 ± 28 μg/ml for A-498 and 769-P cells, respectively. This is the first report showing that green tea is likely to be an effective anticancer agent for renal cell carcinoma.     

Classification of extra virgin olive oils according to their genetic variety using linear discriminant analysis of sterol profiles established by ultra-performance liquid chromatography with mass spectrometry detection

A method to classify extra virgin olive oils (EVOOs) according to their genetic variety using sterol profiles obtained by ultra-performance liquid chromatography (UPLC) with atmospheric pressure chemical ionization mass spectrometry (APCI-MS) detection has been developed. The optimal separation conditions were obtained using a gradient acetonitrile/water (0.01% acetic acid) at a flow rate of 0.8 mL min^− 1 and 10 °C. Linear discriminant analysis (LDA) models were constructed with the 11 UPLC-APCI-MS sterol peaks taken from the selective ion recording mode chromatograms. Ratios of the peak areas selected by pairs were used as predictors. With the sequential application of two LDA models and using a 95% probability, the EVOO samples belonging to seven genetic varieties mainly produced at La Comunitat Valenciana, Spain, were correctly classified with a prediction capability higher than 97%.     

Validation of antibiotics in catfish by on-line solid phase extraction coupled to liquid chromatography tandem mass spectrometry

For the first time automated on-line solid phase extraction coupled to liquid chromatography tandem mass spectrometry was developed for the simultaneous determination of 13 antibiotics (sulfonamides and tetracyclines) in catfish. The method proposed was validated according to Commission Decision 2002/657/EC, showing good linearity between 2 and 350 μg kg⁻¹, high recovery (80–99%) and reproducibility (13–20%) values, lower detection limits than 0.1 μg kg⁻¹, and quantification limits under 2.4 μg kg⁻¹ (between 39 and 84 times lower than the MRL fixed by the EU). Moreover, the proposed method was also used to determine sulfonamides and tetracyclines in 16 out of 107 samples, all previously analysed by microbiological screening that gave positive results. Five out of 13 antibiotics were found, having tetracycline the higher occurrence (10 samples); in all cases the concentrations were lower than the MRL established.     

Determination of selected pesticides in fruit juices by matrix solid-phase dispersion and liquid chromatography–tandem mass spectrometry

A rapid and sensitive liquid chromatography–tandem mass spectrometry method has been developed for the analysis of acephate, monocrotophos, carbendazim, acetamiprid, dimethoate, simazine, carbofuran, atrazine, diuron, DNOC (4,6-dinitro-o-cresol), malathion and tebufenozide in fruit juices. Extracts were obtained by matrix solid-phase dispersion using diatomaceous earth as dispersant and dichloromethane as eluent. Significant matrix effects observed for most of the pesticides tested were eliminated using matrix-matched standards. The quantification of the analytes was carried out using the most sensitive transition. The confirmation of residues detected in real samples was performed by repeated injection and acquiring additional transitions to that used for quantification. Recoveries were in the range 71–118%. Repeatability of the method, expressed as the relative standard deviation, was in general between 5–15%. Low limits of detection (0.01–0.94 ng ml⁻¹) and quantification (0.03–3.12 ng ml⁻¹) were readily achieved with this method for all tested pesticides.