Fatty acids analysis by capillary electrophoresis: Fundamentals, advantages and applications.

This review covers the know-how of the Grupo de Química Analítica e Quimiometria regarding the analysis of fatty acids by capillary electrophoresis acquired over its 20 years of existence. Therefore, the fundamentals, advantages, and applications of this technique for analyzing different fatty acids in samples such as food, oils, cosmetics, and biological matrices are presented and discussed. Capillary electrophoresis is, thus, shown as an interesting and valuable separation technique for the target analysis of these analytes as an alternative to the gas chromatography coupled to flame ionization detection, as it offers advantages over the latter such as low analysis times, low sample and reagent consumption, the use of a nondedicated column, and simpler sample preparation. In addition, the methods shown in this literature review can be useful for quality control, adulteration, and health-related research by regulatory agencies.

A capillary electrophoresis method for free fatty acids screening and acidity determination in biodiesel.

The advent of policies that incentivize or require alternative diesel fuels has increased the demand for the development of fast analytical methods aiming for the quality control of these fuels. This study approached an alternative method for the determination of biodiesel acidity employing capillary zone electrophoresis based on free fatty acids screening and quantification. Sample preparation comprised vortex-assisted liquid-liquid extraction of free fatty acids and was a crucial step for analysis. It was studied through a 32 full factorial design considering sample mass and the stirring time. Then, solvent suitability was evaluated univariately. The free fatty acid screening was carried out employing a capillary zone electrophoresis method able to separate C16:0, C18:0, C18:1 n-9, C18:2 n-6, and C18:3 n-3, major fatty acids in a variety of vegetable oils used for biodiesel synthesis. In addition to the straightforward sample preparation protocol, the running time of the developed method was only 12 min. Moreover, ultraviolet absorption indirect detection of analytes was approached to avoid analytes derivatization, considering the lack of chromophore groups in saturated fatty acids. Statistical tests did not evidence any significant differences in the biodiesel acidity determination expressed in percentage of free fatty acids when comparing the proposed capillary zone electrophoresis method and the traditional potentiometric titration approach within the 95% confidence interval, which demonstrates the suitability of this alternative method for the biodiesel quality control in routine.

Capillary electromigration methods for fatty acids determination in vegetable and marine oils: A review.

Fatty acids determination is of paramount importance for quality control and suitable labeling of edible oils, required by regulatory agencies in several countries, and fast methods for this determination are worldly desired. This review article aimed to explore the available analytical methods for vegetable and marine oils analyses employing CE, which can be a straightforward and faster alternative than GC methods for fatty acid determination, considering some purposes. CE usually offers the possibility of a rapid analysis with a simple preparation of the sample, without requiring specific columns, which are inherent advantages of the technique. Instrumental conditions and the key points about fatty acids determination employing the technique are highlighted, and the main challenges and perspectives are also approached. Potential use of CE for edible oil analyses has been demonstrated for research and routine, which can be of interest for industries, regulatory agencies, and edible oil researchers. Therefore, we have explored the analytical approaches described in the last decades, intending to spread the interest of CE methods for fatty acid monitoring, label accuracy assessment, and food authenticity evaluation of edible oils.

Effects of enzymatic lactose hydrolysis on thermal markers in lactose-free UHT milk.

Lactose-free products are more susceptible to chemical and physical modifications during heating and storage, due to the release of glucose and galactose during enzymatic processing, both more reactive than lactose. The present study demonstrates the effect of enzymatic lactose hydrolysis on 5-hydroxymethylfurfural (HMF), whey protein nitrogen index (WPNI) and lactulose used as thermal markers for UHT milk process monitoring. Six milk leading brands which provided regular and lactose-free UHT milk were selected, giving a total of 12 UHT milk samples analyzed in authentic duplicates. All lactose-free samples showed high levels of HMF index (42.15 µmol L-1, against 13.11 µmol L-1 for regular samples) and low lactulose contents (13.03 mg 100 mL-1, against 35.59 mg 100 mL-1 of regular ones). High variations in HMF (55-85%) and lactulose (42-91%) intra-brand analysis indicated that both markers are influenced by the lactose hydrolysis process. The paired t test indicated there was no difference among WPNI indexes of regular and lactose-free milks suggesting that this thermal marker is suitable to infer about heat damage in lactose-free dairy matrices.

Capillary electrophoresis in association with chemometrics approach for bitterness hop (Humulus lupulus L.) classification.

The precursor compounds related to the bitterness of beer are called α-acids. These compounds are extracted from the hop, which is an important ingredient in the brewing process. These compounds were analyzed by capillary electrophoresis. The electrophoretic method used 160 mmol/L of ammonium carbonate (pH 9) as BGE (background electrolyte), a voltage of +20 kV in a capillary with 50 µm of internal diameter and with a 62.5 cm of total length (54 cm effective). The samples were injected in hydrodynamic mode applying a pressure of 25 mbar for 5 s and the analytes were detected at 230 nm. A hydromethanolic extraction during 3 h was considered as the optimum condition for the sample preparation using MeOH/H2 O 80:20 v/v as the extract solution. From the optimized conditions the electropherograms were evaluated for their use as input for chemometric modeling. Preprocessing investigation for electrophoretic data taking into account the alignment, denoising and baseline correction, and variable selection were considered before the chemometric modeling using principal component analysis (PCA). The electrophoretic data were systematically evaluated to find the optimum conditions to modeling. A PCA analysis for all tests was carried out using different preprocessing methods and, an explained variance higher than 90% was achieved in all of them. The optimized chemometric method worked with aligned and meancentered data. From this approach, a simple and efficient method to classify hop samples with high and low α-acids content without the use of analytical standards was established from a simple electrophoretic analysis.

Sub-minute determination of rifampicin and isoniazid in fixed dose combination tablets by capillary zone electrophoresis with ultraviolet absorption detection.

A validated sub minute capillary zone electrophoresis method with direct ultraviolet absorption detection for simultaneous determination of isoniazid and rifampicin in fixed-dose combination tablets was developed. Background electrolyte was defined based on the analytes effective mobility curve and it was composed by 20 mmol/L of sodium carbonate/sodium bicarbonate at pH 10.2. A careful validation procedure considering the main figures of merit was performed. Regression models were satisfactory for isoniazid and rifampicin, showing no lack of fit within 95% significance interval. Interday and intraday precision were evaluated in standard and sample and slight relative standard deviations were achieved for concentration, area, and migration time. Recovery values for accuracy in two levels were 99.97 and 90.08% for isoniazid and 95.45 and 95.12% for rifampicin. The limits of detection for isoniazid and rifampicin were 0.22 and 0.34 mg/L, respectively, and the limits of quantification were 0.74 and 1.13 mg/L, respectively. Method selectivity was verified by injecting diluent, background electrolyte, a standard mixture, and a sample, confirming no interferent peaks. The method proved to be simple, environmentally friendly, sensitive, and was successfully applied for simultaneous quantification of isoniazid and rifampicin in fixed-dose combination tablets.

Lipid Characterization of White, Dark, and Milk Chocolates by FT-Raman Spectroscopy and Capillary Zone Electrophoresis.

There are few studies about different types of chocolate and their chemical characterization by Fourier transform (FT)-Raman spectroscopy and capillary zone electrophoresis (CZE). The aim of this study was to evaluate the lipid profile of different types of Brazilian chocolate through characterization by FT-Raman spectroscopy and identification and quantification of major fatty acids (FAs) by CZE to confirm FT-Raman spectrometry results. It was found that the main spectroscopic profile difference of the chocolate samples analyzed was related to the presence of saturated or unsaturated FAs. Well defined bands at approximately 1660, 1267, and 1274 cm(-1) corresponding to vibrational modes of unsaturated FAs (UnFAs) were found only in the spectra of samples with cocoa butter in their composition according to label specifications, mainly in dark chocolate samples. The FA identification and quantification by CZE found the presence of stearic (18:0) and palmitic (16:0) acids as the major saturated FAs in all chocolate samples. Dark chocolate samples showed the highest levels of oleic (cis-9 18:1) and linoleic (cis, cis -9,12 18:2) UnFAs monitored and the lowest levels of 14:0 in their chemical composition. Samples coded as 02 (with not only cocoa butter in their composition according to label) had the highest levels of 14:0 (FA not present in cocoa butter composition) corresponding to label information and inferring the presence of other fat sources, called cocoa butter substitutes, mainly for milk and white chocolate samples. This study suggests FT-Raman spectroscopy is a powerful technique that can be used to chemically characterize the chocolate lipid fraction, and CZE is a tool able to confirm Raman spectroscopy results and identify and quantify the major FAs in chocolate samples.

A Rapid Method for Analysis of Phenylalanine in Cereal Products by MEKC-UV Using LC/MS/MS as a Comparative Method.

The aim of this study was to develop a new and fast micellar electrokinetic chromatography (MEKC) method for the determination of phenylalanine in cereal samples. The background electrolyte was chosen by a factorial design and was composed of 30 mmol/L phosphoric acid, 100 mmol/L sodium dodecyl sulfate, and 25% methanol (v/v) at pH 1.9. A fused silica capillary (48.5 cm total length×8.5 cm effective length×50 µm id×375 µm od) was used in a short-end injection configuration, and direct UV detection was at 200 nm. The method was validated following the Eurachem guidelines, and values such as linearity (from 10.1 to 40.4 mg/L); recovery (86.8-103.9%); repeatability (0.06-0.22% for migration time and 1.14-4.82% for peak area); reproducibility (0.04-0.61% for migration time and 2.22-5.72% for peak area); and LOD and LOQ of 20 and 60 mg/100 g, respectively, were obtained. After the comparison involving selectivity and accuracy between capillary electrophoresis and LC/MS/MS method, the MEKC-UV method was successfully applied to analysis of phenylalanine in different cereal products.

Rapid separation of free fatty acids in vegetable oils by capillary zone electrophoresis.

INTRODUCTION: Olive oil is a very important product to human health since it inhibits formation of free radicals, tumour growth, lesions and inflammatory substances. High concentrations of free fatty acids in olive oils results in lipid deterioration due to oxidative or hydrolytic rancidity. OBJECTIVE: To optimise an alternative capillary zone electrophoresis methodology, under ultraviolet indirect detection and to determine free fatty acids in edible vegetable oils without derivatisation steps in sample preparation. METHODS: The condition used consisted of 15 mm NaH2 PO4 -Na2 HPO4 at pH ~6.86, 4.0 mm of sodium dodecybenzenesulphonate, 8.3 mm of Brij 35®, 45% v/v of acetonitrile and 2.1% of 1-octanol, injection at 12.0 mbar of pressure for 4 s, +19 kV of applied voltage and indirect detection at 224 nm, within an analysis time of 11 min. RESULTS: The capillary zone electrophoresis method was successfully applied to determination of free fatty acids in extra virgin olive oil, virgin olive oil and soybean oil samples. The comparison with the official volumetric titration method showed no significant difference within the 95% confidence interval. CONCLUSION: The main advantage to the proposed method is the possibility to observe the individual amount of the free fatty acids, which would be useful for researchers interested in studying the effect of the free fatty acids profile on oxidative process in food.

20 years of fatty acid analysis by capillary electrophoresis.

A review taking into account the literature reports covering 20 years of fatty acid analysis by capillary electrophoresis is presented. This paper describes the evolution of fatty acid analysis using different CE modes such as capillary zone electrophoresis, non-aqueous capillary electrophoresis, micellar electrokinetic capillary chromatography and microemulsion electrokinetic chromatography employing different detection systems, such as ultraviolet-visible, capacitively coupled contactless conductivity, laser-induced fluorescence and mass spectrometry. In summary, the present review signals that CE seems to be an interesting analytical separation technique that is very useful for screening analysis or quantification of the usual fatty acids present in different matrices, offering short analysis times and a simple sample preparation step as inherent advantages in comparison with the classical methodology, making it a separation technique that is very attractive for quality control in industry and government agencies.

Determination of creatinine in urine and blood serum human samples by CZE-UV using on-column internal standard injection.

Creatinine is a well-stablished biomarker for kidney malfunctions and for normalization parameter of urinary quantitative information. Recently, metabolic studies have been discovering other functionalities for creatinine tests in human urine and blood serum. In this work we present an enhanced capillary electrophoresis (CE) based protocol for determination of creatinine. CE is a high-throughput separation technique that have been getting attention through the last decades and might be considered to be adopted as an analytical instrumentation for clinical purposes. In the proposed method, we performed a short injection program with on-column addition of internal standard. Additionally, the method allows a simultaneous screening of non-proteinogenic amino acids that could be considered for metabolomics purposes. We design a pilot study that successfully estimated the creatinine value in 100 urine samples with (2.85 ± 1.78) mg dL-1 LOD; (8.24 ± 5.93) mg dL-1 LOQ and 82.4% accuracy. Considering that serum creatinine is also included in the clinical laboratory routines for estimated Glomerular Filtration Rate dosage, the method was complementary applied to 10 blood serum samples, which resulted in a model with (0.4 ± 0.2) mg dL-1 LOD; (2.0 ± 0.6) mg dL-1 LOQ and 83.8% of accuracy. All results were in agreement with reference values. The proposed method promotes a great analytical frequency and reproducibility with enhanced specificity compared with the ongoing protocol by Jaffe's reaction, thereby proving to be useful as an alternative for creatinine exams that might help complete a diagnosis of a series of health-related issues.

Optimisation of a capillary zone electrophoresis methodology for simultaneous analysis of organic aliphatic acids in extracts of Brachiaria brizantha.

INTRODUCTION: Aluminum toxicity is commonly verified in acidic soils, and poses a severe limitation to plant growth and development. Therefore, Al complexation by the root system mucilage, Al complexation by organic compounds that are exuded by the roots and internal metabolic processes must be monitored by organic acids (OA), since they play a central role in these aluminum tolerance mechanisms. OBJECTIVE: To optimise a capillary zone electrophoresis method able to perform simultaneous separation of acetic, citric, formic, lactic, malic, oxalic, pyruvic, succinic, tartaric and aspartic acid in plant extract solutions. METHODOLOGY: Method optimisation was achieved by a chemometric approach through experimental designs. The optimal condition found was: 20 mmol/L phthalic acid buffer; 0.8 mmol/L cetyltrimethyl-ammonium bromide; pH 3.4 adjusted with tris(hydroxymethyl)aminomethane (around 16 mmol/L); -15 kV of voltage; 25 °C of cartridge temperature; indirect ultraviolet detection at 240 nm; and 25 mbar injection for 2 s, within an analysis time of 4 min. RESULTS: As a repeatability test of the optimal condition, 30 replicates were carried out with the same working electrolyte, where the relative standard deviation of each peak ranged from 0.081 to 0.36% (for migration time) and from 2.4 to 4.6% (for peak area). CONCLUSION: The methodology was successfully applied to simultaneously determine citric, malic and aspartic acid in roots and leaves extract solutions of Brachiaria brizantha, demonstrating its usefulness to study aluminum tolerance.

Origin geographical classification of green coffee beans (Coffea a rabica L.) produced in different regions of the Minas Gerais state by FT-MIR and chemometric.

The present work was proposal the potential evaluation of Fourier-Transform Mid-Infrared (FT-MIR) associated with chemometric approach in green beans, in order to discriminate the origin of special Arabica coffees in a single state that has heterogeneous environments. Partial Least Squares Discriminant Analysis (PLS-DA) model presented as result: 3 latent variables, R 2 X (cum) = 0.892, R 2 Y (cum) = 0.659; Q 2 Y (cum) = 0.494, RMSEP = 0.182387, p-value CV-Anova = 0.009, 100% of both sensitivity and specificity and the prediction classification obtained was: 100, 83.33, 100, 83.33% for class 1, class 2, class 3 and class 4, respectively. These results can be considered adequate for the proposed hypothesis. The obtained results that the regions have markers such as trigonelline, chlorogenic and fatty acids, sensitive to absorption in the mid-infrared and that are able to determine the origin of green coffee beans of Arabica. Thus, the FT-MIR associated with chemometrics has the potential to employ speed, modernity and cost reduction in the certification of origin of coffees.

Determination of purity and anionic exchange efficiency of amino acid ionic liquids synthesis by multiple-injection capillary zone electrophoresis.

The purity of ionic liquids (IL) is important for its performance, so the methods able to quantify low concentration impurities are necessaries. Therefore, this paper aims to determine the purity and the anionic exchange efficiency for the preparation of amino acids ionic liquids (AAIL). For this, a Multiple-Injection Capillary Zone Electrophoresis (MICZE) method with direct UV detection was developed and optimized for iodide (I-) determination as impurity of AAIL synthesis, comparing this method with traditional injection modes. Furthermore, this paper aims to demonstrate the use of factorial design methods for the optimization of MICZE method. The method optimization allowed five consecutives I- injections of sample in a single run, with analysis time of less than 3 min, showing a larger analytical frequency, counting with 31 injections per hour. It was possible to determine the high purity of the prepared and analyzed AAIL, ranging from 89% to 95% and its overall ionic exchange yield varying from 55% to 87%.

Determination of α- and ß-acids in hops by liquid chromatography or electromigration techniques: A critical review.

Hop plays an essential role in brewing beer and its study and analysis is of paramount importance. - and -acids are considered two of the most important hop components. While -acids are associated with the bitter flavor, -acids have antimicrobial effects. This work aims to critically review the published analytical methods for - and -acids determination in hops employing separation methods in liquid medium: liquid chromatography (LC) and capillary electrophoresis (CE). The types of hop samples, the optimized protocols to extract the hop acids, and the main instrumental conditions for both LC and CE techniques are highlighted and discussed. Specific and critical aspects of the - and ß-acids separation by LC and CE and some challenges in this field are raised. Several key aspects discussed in this review may be of practical importance for brewers, whether in the microbrewery or industry and for researchers in the brewing field.

Effects of Avocado Oil Supplementation on Insulin Sensitivity, Cognition, and Inflammatory and Oxidative Stress Markers in Different Tissues of Diet-Induced Obese Mice.

Obesity induces insulin resistance, chronic inflammation, oxidative stress, and neurocognitive impairment. Avocado oil (AO) has antioxidants and anti-inflammatory effects. This study evaluated the effect of AO supplementation on obese mice in the adipose tissue, muscle, liver, and hippocampus. Male C57BL/6J mice received a standard and high-fat diet (20 weeks) and then were supplemented with AO (4 mL/kg of body weight, 90 days) and divided into the following groups: control (control), control + avocado oil (control + AO), diet-induced obesity (DIO), and diet-induced obesity + avocado oil (DIO + AO) (n = 10/group). AO supplementation was found to improve insulin sensitivity and decrease hepatic fat accumulation and serum triglyceride levels in DIO mice. AO improved cognitive performance and did not affect mood parameters. Oxidative marker levels were decreased in DIO + AO mice in all the tissues and were concomitant with increased catalase and superoxide dismutase activities in the epididymal adipose tissue and quadriceps, as well as increased catalase activity in the liver. AO in obese animals further induced reductions in TNF-α and IL-1ß expressions in the epididymal adipose tissue and quadriceps. These results suggest that AO supplementation has the potential to be an effective strategy for combating the effects of obesity in rats, and human studies are needed to confirm these findings.

Short-End Injection Capillary Electrophoresis and Multivariate Analysis for Simultaneous Determination of Heavy Metals in Passiflora incarnata Tea.

An ultra-fast method for the simultaneous determination of heavy metals in Passiflora incarnata tea by capillary electrophoresis (CE) using a short-end injection combined with multivariate analysis was proposed. Separation was conducted by hydrodynamic injection (5 s at 0.5 psi) using the short-end injection procedure in a fused uncoated silica capillary (50 cm total length, 10.2 cm effective length, 50 µm i.d.) with separation time less than 2 min. An indirect UV detection at 214 nm was employed by using imidazole as a chromophore. The buffer used was 6 mmol/L hydroxybutyric acid (HIBA). The optimum conditions by full factorial with a central point were achieved by 18-crown-6 concentration (23.3 mmol L−1), voltage (+11.4 kV), methanol concentration (3.8%), and temperature (20 °C). The method showed good linearity (R2 > 0.998) for both Cd and Pb, inter-day precision of less than 14.49%, and an adequate limit of quantification only for Cd (LOQ < 0.5 µg mL−1 for Cd) based on the US Pharmacopeial Convention limit requirements for elemental impurities. After method validation, the method was applied to Passiflora incarnata tea samples from a local market. Furthermore, the developed method showed great potential for the determination of metals in other samples with proper sample preparation procedures.

Determination of hydroquinone and benzoquinone in pharmaceutical formulations: critical considerations on quantitative analysis of easily oxidized compounds.

Hydroquinone is a skin-lightening agent used as an active ingredient in topical dermatological formulations prescribed for treating cutaneous diseases caused by hyperpigmentation. Despite being widely used, some toxicological aspects have been associated with these products, mainly due to overdosage and long-term use combined with the easy oxidation of hydroquinone. In this work, an investigative study has been done to gather enough data for selecting a quantitative analytical method for quality control purposes that considers the ease of oxidation not only within the product but also during the experimental procedures. After studying the influence of pH, reversibility, sampling, and standard solution preparation on the redox reaction between hydroquinone and benzoquinone by using spectroscopic, electrophoretic, and electroanalytical measurements, a reliable, fast, and selective chronoamperometric method was achieved. The optimized method was used for the analysis of samples, previously diluted in Britton-Robinson (BR) buffer (pH 5.5) and methanol (1 : 9, v/v), by applying a potential fixed at 0.4 V. A glassy-carbon working electrode, lab-made Ag/AgCl(sat) and platinum wire as a reference electrode and auxiliary electrodes, respectively, and BR buffer (pH 5.5) as supporting electrolyte were the additional experimental conditions used. Analytical performance parameters were verified to confirm the applicability of the new method (LOD 4.22 µmol L-1 and LOQ 14.1 µmol L-1; recovery mean value of 100% with 0.22% RSD). A gel topical formulation containing 4% (w/w) hydroquinone was analyzed through the developed method for determination of dosage and oxidation traces, and a content of 3.53 ± 0.095% (w/w) was found with no indications of degradation.

Determination of antimalarial drugs in pharmaceutical formulations and human blood by liquid chromatography: a review.

Malaria is a life-threatening disease being treated by oral medication. This is the best treatment to reduce morbidity and mortality, prevent disease progression to the most severe form, lower the transmission of the disease and hinder the appearance of strains resistant to antimalarials. According to the World Health Organization, the most common antimalarial drugs are chloroquine, primaquine, mefloquine, lumefantrine, artemether, and artesunate in single dosage forms or fixed-dose combination. Within this context, the present review aims to show the evolution of different analytical methods that have been applied to the determination of these antimalarial drugs in pharmaceutical formulations and human blood by liquid chromatography in the last 10 years, along with statistical analyses of the methods.

Fast determination of ethambutol in pharmaceutical formulations using capillary electrophoresis with capacitively coupled contactless conductivity detection.

A method for the determination of ethambutol (EMB), a first-line drug against tuberculosis, based on CE with capacitively coupled contactless conductivity detection is proposed. The separation of EMB and its main product of degradation were achieved in less than 3 min with a resolution of 2.0 using a BGE composed of 50 mmol/L histidine and 30 mmol/L MES, pH 6.30. By raising the pH to 8.03, the analysis time was reduced to 1.0 min, but with a significant loss of resolution (0.7). Using the best separation conditions, linearity of 0.9976 (R(2), five data points), sensitivity of 1.26x10(-4) V min mumol(-1) L, and LOD and quantification of 23.5 and 78.3 mumol/L, respectively, were obtained. Recoveries at four levels of concentration ranged from 95 to 102% and the concentration range studied ranged from 100 to 500 mumol/L. The results obtained for the determination of EMB in pharmaceutical formulations were compared with those obtained by using CE with photometric detection.