Determination of eight fatty acid ethyl esters in meconium samples by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A number of fatty acid ethyl esters (FAEEs) have recently been detected in meconium samples. Several of these FAEEs have been evaluated as possible biomarkers for in utero ethanol exposure. In the present study, a method was optimized and validated for the simultaneous determination of eight FAEEs (ethyl laurate, ethyl myristate, ethyl palmitate, ethyl palmitoleate, ethyl stearate, ethyl oleate, ethyl linoleate and ethyl arachidonate) in meconium samples. FAEEs were extracted by headspace solid-phase microextraction. Analyte detection and quantification were carried out using GC-MS operated in chemical ionization mode. The corresponding D5-ethyl esters were synthesized and used as internal standards. The LOQ and LOD for each analyte were <150 and <100 ng/g, respectively. The method showed good linearity (r(2)>0.98) in the concentration range studied (LOQ -2000 ng/g). The intra- and interday imprecision, given by the RSD of the method, was lower than 15% for all FAEEs studied. The validated method was applied to 63 authentic specimens. FAEEs could be detected in alcohol-exposed newborns ( >600 ng/g cumulative concentration). Interestingly, FAEEs could also be detected in some non-exposed newborns, although the concentrations were much lower than those measured in exposed cases.

High Throughput Simultaneous Assay of Atenolol and Chlortalidone in Combined Dose Tablets by Liquid Chromatography and Capillary Zone Electrophoresis

New fast liquid chromatographic and capillary zone electrophoresis methods were developed and validated for simultaneous determination of atenolol and chlortalidone in combined dose tablets. The reversed phase HPLC method was carried out on a CN LiChrosorb (R) (125 x 4 mm, 5 mu m) column. The CZE method was carried out on an uncoated fused-silica capillary of 30 cm x 75 mu m i.d. with 25 mmol L(-1) sodium tetraborate, pH 9.4. The total analysis time was <6 and <2.5 min for HPLC and CZE methods, respectively. Both methods can be used for stability studies as well.

Simultaneous analysis of parabens in cosmetic products by stir bar sorptive extraction and liquid chromatography

A sensitive and precise stir bar sorptive extraction (SBSE) combined with LC (SBSE/LC) analysis is described for simultaneous determination of methyl, ethyl, propyl, and butyl parabens in commercial cosmetic products in agreement with the European Union Cosmetics Directive 76/768/EEC. Important factors in the optimization of SB SE efficiency are discussed, such as time and temperature of extraction, pH, and ionic strength of the sample, matrix effects, and liquid desorption conditions by different modes (magnetic stirring, ultrasonic). The LOQs of the SBSE/LC method ranged from 30 to 200 ng/mg, with linear response over a dynamic range, from the LOQ to 2.5 mu g/mg, with a coefficient of determination higher than 0.993. The interday precision of the SBSE/LC method presented a coefficient of variation lower than 5%. The effectiveness of the proposed method was proven for analysis of commercial cosmetic products such as body creams, antiperspirant creams, and sunscreens.

Determination of carbamate and urea pesticide residues in fresh vegetables using microwave-assisted extraction and liquid chromatography

An analytical multiresidue method for the simultaneous determination of seven pesticides in fresh vegetable samples, namely, courgette (Cucurbita pepo), cucumber (Cucumis sativus), lettuce (Lactuca sativa, Romaine and Iceberg varieties) and peppers (Capsicum sp.) is described. The procedure, based on microwave-assisted extraction (MAE) and analysis by liquid chromatography– photodiode array (LC–PDA) detection was applied to four carbamates (carbofuran, carbaryl, chlorpropham and EPTC) and three urea pesticides (monolinuron, metobromuron and linuron). Extraction solvent and the addition of anhydrous sodium sulphate to fresh vegetable homogenate before MAE were the parameters optimised for each commodity. Recovery studies were performed using spiked samples in the range 250–403 µgkg- 1 in each pesticide. The pesticide residues were extracted using 20mL acetonitrile at 60 ºC, for 10 min. Acceptable recoveries and RSDs were attained (overall average recovery of 77.2% and RSDs are lower than 11%). Detection limits ranged between 5.8 µgkg- 1 for carbaryl to 12.3 µgkg- 1 for carbofuran. The analytical protocol was applied for quality control of 41 fresh vegetable samples bought in Oporto Metropolitan Area (North Portugal). None of the samples contained any detectable amounts of the studied compounds.

Optimization of QuEChERS procedure coupled to GC-ECD for organochlorine pesticide determination in carrot samples

An optimised version of the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method for simultaneous determination of 14 organochlorine pesticides in carrots was developed using gas chromatography coupled with electron-capture detector (GC-ECD) and confirmation by gas chromatography tandem mass spectrometry (GC-MS/MS). A citrate-buffered version of QuEChERS was applied for the extraction of the organochlorine pesticides, and for the extract clean-up, primary secondary amine, octadecyl-bonded silica (C18), magnesium sulphate (MgSO4) and graphitized carbon black were used as sorbents. The GC-ECD determination of the target compounds was achieved in less than 20 min. The limits of detection were below the EUmaximum residue limits (MRLs) for carrots, 10–50 μg kg−1, while the limit of quantification did exceed 10 μg kg−1 for hexachlorobenzene (HCB). The introduction of a sonication step was shown to improve the recoveries. The overall average recoveries in carrots, at the four tested levels (60, 80, 100 and 140 μg kg−1), ranged from 66 to 111% with relative standard deviations in the range of 2– 15 % (n03) for all analytes, with the exception of HCB. The method has been applied to the analysis of 21 carrot samples from different Portuguese regions, and β-HCH was the pesticide most frequently found, with concentrations oscillating between less than the limit of quantification to 14.6 μg kg−1. Only one sample had a pesticide residue (β-HCH) above the MRL, 14.6 μg kg−1. This methodology combines the advantages of both QuEChERS and GC-ECD, producing a very rapid, sensitive and reliable procedure which can be applied in routine analytical laboratories.

SPE-LC-FD Determination of Polycyclic Aromatic Hydrocarbon Monohydroxy Derivatives in Cephalopods

A new analytical methodology, based on liquid chromatography with fluorescence detection (LC-FD), after extraction, enzymatic hydrolysis, and solid-phase extraction (SPE) through Oasis HLB cartridges, was developed and validated for the simultaneous determination of three monohydroxy derivatives of polycyclic aromatic hydrocarbons (PAHs). The optimized analytical method is sensitive, accurate, and precise, with recoveries between 62 and 110% and limits of detection of 227, 9, and 45 ng/g for 1-hydroxynaphthalene, 2-hydroxyfluorene, and 1-hydroxypyrene, respectively. Their levels were estimated in different cephalopod matrices (edible tissues and hemolymph). The methodology was applied to samples of the major cephalopod species consumed worldwide. Of the 18 samples analyzed, 39% were contaminated with 1-hydroxynaphthalene, which was the only PAH metabolite detected. Its concentration ranged from 786 to 1145 ng/g. This highly sensitive and specific method allows the identification and quantitation of PAH metabolites in forthcoming food safety and environmental monitoring programs.

Simultaneous Evaluation of Intact Glucosinolates and Phenolic Compounds by UPLC-DAD-MS/MS in Brassica oleracea L. var. botrytis

A method for the simultaneous determination of intact glucosinolates and main phenolic compounds (flavonoids and sinapic acid derivatives) in Brassica oleracea L. var. botrytis was proposed. A simplified sample extraction procedure and a UPLC separation were carried out to reduce the total time of analysis. Brassica oleracea samples were added with internal standards (glucotropaeolin and rutin), and extracted with boiling methanol. Crude extracts were evaporated under nitrogen, redissolved in mobile phase and analyzed by UPLC with double detection (ESI--MRM for glucosinolates and flavonoids, and DAD for main sinapic acid derivatives). The proposed method allowed a satisfactory quantification of main native sinapic acid derivatives, flavonoids and glucosinolates with a reduced time of analysis.

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity

Multiplex liquid chromatography-tandem mass spectrometry assay for simultaneous therapeutic drug monitoring of ribavirin, boceprevir, and telaprevir.

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Complexometric titration with potenciometric indicator to determination of calcium and magnesium in soil extracts¹

This study proposes a method of direct and simultaneous determination of the amount of Ca2+ and Mg2+ present in soil extracts using a Calcium Ion-Selective Electrode and by Complexometric Titration (ISE-CT). The results were compared to those obtained by conventional analytical techniques of Complexometric Titration (CT) and Flame Atomic Absorption Spectrometry (FAAS). There were no significant differences in the determination of Ca2+ and Mg2+ in comparison with CT and FAAS, at a 95 % confidence level. Additionally, results of this method were more precise and accurate than of the Interlaboratorial Control (IC).

Sensitive determination of D-lactic acid and L-lactic acid in urine by high-performance liquid chromatography-tandem mass spectrometry.

D-lactic acid in urine originates mainly from bacterial production in the intestinal tract. Increased D-lactate excretion as observed in patients affected by short bowel syndrome or necrotizing enterocolitis reflects D-lactic overproduction. Therefore, there is a need for a reliable and sensitive method able to detect D-lactic acid even at subclinical elevation levels. A new and highly sensitive method for the simultaneous determination of L- and D-lactic acid by a two-step procedure has been developed. This method is based on the concentration of lactic acid enantiomers from urine by supported liquid extraction followed by high-performance liquid chromatography-tandem mass spectrometry. The separation was achieved by the use of an Astec Chirobiotic? R chiral column under isocratic conditions. The calibration curves were linear over the ranges of 2-400 and 0.5-100 µmol/L respectively for L- and D-lactic acid. The limit of detection of D-lactic acid was 0.125 µmol/L and its limit of quantification was 0.5 µmol/L. The overall accuracy and precision were well within 10% of the nominal values. The developed method is suitable for production of reference values in children and could be applied for accurate routine analysis.

Comparison of three analytical methods for the determination of quinolones in pig muscle samples

This work presents a comparison between three analytical methods developed for the simultaneous determination of eight quinolones regulated by the European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid and flumequine) in pig muscle, using liquid chromatography with fluorescence detection (LC-FD), liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedures involve an extraction of the quinolones from the tissues, a step for clean-up and preconcentration of the analytes by solid-phase extraction and a subsequent liquid chromatographic analysis. The limits of detection of the methods ranged from 0.1 to 2.1 ng g−1 using LC-FD, from 0.3 to 1.8 using LC-MS and from 0.2 to 0.3 using LC-MS/MS, while inter- and intra-day variability was under 15 % in all cases. Most of those data are notably lower than the maximum residue limits established by the European Union for quinolones in pig tissues. The methods have been applied for the determination of quinolones in six different commercial pig muscle samples purchased in different supermarkets located in the city of Granada (south-east Spain).

UV determination of epinephrine, uric acid, and acetaminophen in pharmaceutical formulations and some human body fluids using multivariate calibration

In this work, a spectrophotometric methodology was applied in order to determine epinephrine (EP), uric acid (UA), and acetaminophen (AC) in pharmaceutical formulations and spiked human serum, plasma, and urine by using a multivariate approach. Multivariate calibration methods such as partial least squares (PLS) methods and its derivates were used to obtain a model for simultaneous determination of EP, UA and AC with good figures of merit and mixture design was in the range of 1.8 - 35.3, 1.7 - 16.8, and 1.5 - 12.1 µg mL-1. The 2nd derivate PLS showed recoveries of 95.3 - 103.3, 93.3 - 104.0, and 94.0 - 105.5 µg mL-1 for EP, UA, and AC, respectively.

Flow-batch analyser for preparation of calibration standard mixtures in simultaneous multicomponent spectrometric analysis

The application of multivariate calibration techniques to multicomponent analysis by UV-VIS molecular absorption spectrometry is a powerful tool for simultaneous determination of several chemical species. However, when this methodology is accomplished manually, it is slow and laborious, consumes high amounts of reagents and samples, is susceptible to contaminations and presents a high operational cost. To overcome these drawbacks, a flow-batch analyser is proposed in this work. This analyser was developed for automatic preparation of standard calibration and test (or validation) mixtures. It was applied to the simultaneous determination of Cu2+, Mn2+ and Zn2+ in polyvitaminic and polymineral pharmaceutical formulations, using 4-(2-piridilazo) resorcinol as reagent and a UV-VIS spectrophotometer with a photodiode array detector. The results obtained with the proposed system are in good agreement with those obtained by flame atomic absorption spectrometry, which was employed as reference method. With the proposed analyser, the preparation of calibration and test mixtures can be accomplished about four hours, while the manual procedure requires at least two days. Moreover, it consumes smaller amounts of reagents and samples than the manual procedure. After the preparation of calibration and test mixtures, 60 samples h-1 can be carried out with the proposed flow-batch analyser.

A method for fast determination of epigallocatechin gallate (EGCG), epicatechin (EC), catechin (C) and caffeine (CAF) in green tea using HPLC

Tea has been considered a medicine and a healthy beverage since ancient times, but recently it has received a great deal of attention because of its antioxidant properties. Green tea polyphenols have demonstrated to be an effective chemopreventive agent. Recently, investigators have found that EGCG, one of the green tea catechins, could have anti-HIV effects when bound to CD4 receptor. Many factors can constitute important influences on the composition of tea, such as species, season, age of the leaf, climate, and horticultural practices (soil, water, minerals, fertilizers). This paper presents an HPLC analytical methodology development, using column RP-18 and mobile phase composed by water, acetonitrile, methanol, ethyl acetate, glacial acetic acid (89:6:1:3:1 v/v/v/v/v) for simultaneous determination and quantification of caffeine (CAF), catechin (C), epicatechin (EC) and epigallocatechin gallate (EGCG) in samples of Camellia sinensis (green tea) grown in Brazil and harvested in spring, in summer and in autumn, in comparison to Brazilian black tea, to samples of Japanese and Chinese green tea and to two standardized dry extracts of green tea. The method has been statistically evaluated and has proved to be adequate to qualitative and quantitative determination of the samples.