A liquid chromatographic method optimization for the assessment of low and high molar mass carbonyl compounds in wines

Carbonyl compounds (CC) play an important role in beverage aroma since they may affect flavor of wines, brandies, and beers, among others. For this reason, it is necessary to identify and quantify CC through adequate analytical techniques. This study is a proposal of both developing and optimization of a new analytical methodology that allows investigate C₁–C₈ CC in wines simultaneously by quantifying even those ones that are predominantly present in the adduct form hydroxylalkylsulfonic acids (HASA). The HASA dissociation is undertaken by specific alkaline media (pH 11). The developed methodology employed the LC with UV/VIS detection (λ = 365 nm) technique under gradient elution in the way to reach both free‐CC and bound‐CC quantification. Results showed that binary gradient system using eluent A (MeOH/ACN/H₂O 74.5:0.5:25% v/v/v) and eluent B (MeOH) reached the best separation condition of both lower and higher molecular mass CC. This proposed method allowed simultaneous quantification of formaldehyde, acetaldehyde, propanone, furfuraldehyde, butyraldehyde, benzaldehyde, hexanaldehyde, 2‐ethyl‐hexanaldehyde, E‐pent‐2‐en‐1‐al, and cyclohexanone – all of them were found in white wine (Moscato Canelli) and red wine (Shiraz) produced in the São Francisco Valley, in the Northeastern Region of Brazil – although this optimized method may probably be suitable for quantification of propionaldehyde, isobutyraldehyde, heptanaldehyde, octanaldehyde, benzaldehyde, and E‐hex‐2‐en‐1‐al as well. We could not prove if this method is also able to determine the latter CC group since we have not found these substances present in detectable levels in our real samples considered in this study.     

Determination of clofentezine in medical herb extracts by chromatographic methods combined with diode array scanning densitometry

The aim of this study is to report a new procedure for extraction, cleanup and determination of clofentezine in herb extracts by ultrasound‐assisted solvent extraction, SPE and multidimensional planar chromatography with diode array detector (MDPC‐DAD) and/or HPLC‐DAD. The application of various extraction solvents in SPE experiments conducted on octadecyl silane coupled with styrene‐divinylbenzene cartridges for fractionation and purification samples has been described. Normal‐phase systems were used in MDPC experiments on silica layer. The procedure described for the determination of compounds is inexpensive and can be applied to the routine analysis of analytes in plant extracts, after preliminary cleanup and concentration, e.g. by SPE. Application of MDPC‐DAD and HPLC‐DAD is especially useful for correct identification of components of difficult, complicated mixtures, e.g. analytes in medical herbs.     

Evaluation of matrix solid‐phase dispersion extraction for the determination of polycyclic aromatic hydrocarbons in household dust with the aid of experimental design and response surface methodology

A simple, fast, and inexpensive procedure for sample preparation based on matrix solid‐phase dispersion was developed for the determination of Environmental Protection Agency 16 priority polycyclic aromatic hydrocarbons in indoor dust samples. Parameters that affect the extraction efficiency such as type of dispersant, elution solvent, and solvent volume were evaluated and optimized with the aid of experimental design and response surface methodology. Analysis was performed by HPLC coupled with UV‐Vis diode array detector (UV‐DAD). For verification, a GC coupled with a mass spectrometer in SIM mode was also applied. Recoveries obtained were from 53 to 120% for all target analytes with detection limits ranging from 0.2 to 10 ng/g and 0.2 to 2 ng/g for LC‐UV‐DAD and GC‐MS, respectively. The optimized method was used for the analysis of 11 household dust samples collected from private houses.     

Sample clean-up by sol-gel immunoaffinity chromatography for determination of chloramphenicol in shrimp

The paper describes the characterisation and application of sol-gel columns prepared by entrapping anti-chloramphenicol (CAP) antibodies. Retention of CAP in the column was caused by specific interactions with the anti-CAP antibodies and not by non-specific adsorption to the sol-gel glass. After optimising important operation conditions, e.g. feeding medium, feeding flow-rate, elution medium, elution flow-rate and elution volume, the sol-gel columns were included in a clean-up procedure developed to determine CAP in shrimp. The selectivity of the columns was high enough to efficiently remove interfering matrix compounds. Due to the chromatographic conditions applied retention of cross-reacting substances in the immunoaffinity column did not pose a problem. CAP recovery of the analytical method was 68% with a relative standard deviation of 4% (n=4). In spite of applying highly complex shrimp extracts the columns could be used for clean-up of at least 12 samples. However, when detection of CAP is carried out with an UV detector the analytical method has a relatively poor sensitivity (LOD=1.8 ng/g, S/N=3). The most obvious way is to replace the UV detector by a detector based on an inherently more sensitive and selective detection principle, like a mass spectrometer.     

Simultaneous determination of catechins, rutin, and gallic acid in Cistus species extracts by HPLC with diode array detection

A simple high-performance liquid chromatography method using a diode array detector (DAD) is developed for the simultaneous analysis of five major catechins: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GCT), (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCG), and the phenolic plant metabolites gallic acid (GA) and rutin (RT) in lyophilized extracts of Cistus species. The optimal analytical conditions are investigated to obtain the best resolution and the highest UV sensitivity for the quantitative detection of catechins. The optimized conditions (acetonitrile-phosphate buffer 50mM, pH 2.5, gradient elution system on a C(18) reversed-phase column with a flow rate of 1 mL/min and UV absorbance at 210 nm) allowed a specific and repeatable separation of the studied analytes to be achieved. All compounds are successfully separated within 32 min. Calibration curves are linear in the 2-50 microg/mL range for GCT, C, and EGCG and in the 5-50 microg/mL range for GA, EGC, EC, and RT. The limit of detection values ranged from 0.24 to 0.74 microg/mL. The limit of quantitation limit values ranged from 0.77 to 1.94 microg/mL. The validated method is applied to the determination of the specific phytochemical markers GA, GCT, C, and RT in Cistus incanus and Cistus monspeliensis lyophilised extracts. The recovery values ranged between 78.7% and 98.2%. The described HPLC method appears suitable for the differentiation and determination of the most common catechins together with the glycoside rutin and the phenolic compound gallic acid and can be considered an effective and alternative procedure for the analyses of this important class of natural compounds.     

Comparison of gas and high performance liquid chromatography with selective detection for determination of triazine herbicides and their degradation products extracted ultrasonically from soil

GC and HPLC with selective detectors were compared for simultaneous determination of triazine herbicides simazine, atrazine, propazine, terbuthylazine, cyanazine, ametryn, prometryn, and atraton, and of their dealkylated degradation products in soil. The compounds were ultrasonically extracted from spiked agricultural soil samples (organic matter content < 5%) with a 2:1 acetone:n‐hexane mixture. High efficiency of GC capillary column and high selectivity of the thermionic sensitive detector (TSD) and ion trap detector (ITD) made it possible to directly analyse uncleaned soil extracts and determine all 12 compounds in one run. In reversed‐phase HPLC with diode‐array detector (DAD), the co‐elution of soil matrix components interfered with the determination of methylthiotriazines and terbuthylazine. The recovery of triazine compounds, determined by GC‐TSD, from a silty sand soil (organic matter content 1.82%, pH 6.22) spiked at levels of 15–600 ng g^–1, were 70–90% (RSD 9–19%), except for deisopropylatraton (38%). GC‐TSD analysis with detection limits of 5–15 ng g^–1 for chloro‐ and methylthiotriazines and 30 ng g^–1 for methoxytriazines was more sensitive than GC‐MS(ITD). GC analysis with electron capture detection was sensitive for some chlorotriazines, but a reliable compound quantification in complex chromatograms of uncleaned soil of extracts was not possible. For all compounds save didealkylatrazine, HPLC‐DAD was at least two times less sensitive than GC‐TSD. Soil/sediment organic matter, clay and silt content, and pH were identified as matrix characteristics which might affect ultrasonic extraction recovery of a particular compound.     

Determination of target fat‐soluble micronutrients in rainbow trout's muscle and liver tissues by liquid chromatography with diode array‐tandem mass spectrometry detection

This paper describes an analytical approach, based on LC‐diode array detector‐MS/MS (LC‐DAD‐MS/MS), for characterizing the fat‐soluble micronutrient fraction in rainbow trout (Oncorhynchus mykiss ). Two different procedures were applied to isolate the analytes from liver and muscle tissue: overnight cold saponification to hydrolyze bound forms and to simplify the analysis; matrix solid‐phase dispersion to avoid artifacts and to maintain unaltered the naturally occurring forms. Analytes were separated on a C₃₀ analytical column by using a nonaqueous reversed mobile phase compatible with the atmospheric pressure chemical ionization. Compared to other works, the most relevant advantage of the here illustrated method is the large amount of information obtained with few analytical steps: nine fat‐soluble vitamins (3,4‐dehydroretinol, retinol, cholecalciferol, ergocalciferol, α‐tocopherol, γ‐tocopherol, δ‐tocopherol, phylloquinone, and menaquinone‐4) and eight carotenoids (all‐trans ‐lutein, all‐trans ‐astaxanthin, all‐trans ‐zeaxanthin, all‐trans ‐β‐cryptoxanthin, all‐trans ‐canthaxanthin, all‐trans ‐ζ‐carotene, all‐trans ‐β‐carotene, and all‐trans ‐γ‐carotene) were quantified after the method validation, while other untargeted carotenoids were tentatively identified by exploiting the identification power of the LC‐DAD‐MS/MS hyphenation.     

HPLC separation and relative quantitation of kaempferol glycosides in soybean

Summary An HPLC method is described for the determination of kaempferol glycosides in soybean leaf extracts. The method is rapid and can provide qualitative and relative quantitative results for 9 kaempferol glycosides. The flavonol glycosides are extracted from soybean leaves by shaking the samples in 50% methanol; the extracts are filtered, evaporated to dryness and reconstituted in methanol before further purification through a C-18 Sep-Pak column. The samples are injected onto a C-18 HPLC column, separated by gradient elution with a 1% phosphoric acid: methanol mixture and detected on a UV/VIS diode-array detector. Flavonols were monitored at 265 and 348 nm and spectra from 180 to 400 nm were stored and used as an aid in identification. Relative quantities of the kaempferol glycosides among soybean cultivars were calculated from their proportion of peak area in the chromatograms. Total kaempferol concentration of the extracts was calculated after acid hydrolysis of the kaempferol glycosides to the aglycone and comparison of peak areas to kaempferol standards.     

Analysis of phenolic compounds from different morphological parts of Helichrysum devium by liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection

A simple and rapid method has been used for the screening and identification of the main phenolic compounds from Helichrysum devium using high‐performance liquid chromatography with on‐line UV and electrospray ionization mass spectrometric detection (LC‐DAD/ESI‐MSⁿ). The total aerial parts and different morphological parts of the plant, namely leaves, flowers and stems, were analyzed separately. A total of 34 compounds present in the methanolic extract from Helichrysum devium were identified or tentatively characterized based on their UV and mass spectra and retention times. Three of these compounds were positively identified by comparison with reference standards. The phenolic compounds included derivatives of quinic acid, O‐glycosylated flavonoids, a caffeic acid derivative and a protocatechuic acid derivative. The characteristic loss of 206 Da from malonylcaffeoyl quinic acid was used to confirm the malonyl linkage to the caffeoyl group. This contribution presents one of the first reports on the analysis of phenolic compounds from Helichrysum devium using LC‐DAD/ESI‐MSⁿ and highlights the prominence of quinic acid derivatives as the main group of phenolic compounds present in these extracts. We also provide evidence that the methanolic extract from the flowers was significantly more complex when compared to that of other morphological parts. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of the major compounds in extracts ofVerbena officinalis L. (Verbenaceae) by HPLC with post-column derivatization

Summary A qualitative reversed-phase HPLC method has been developed for the analysis of 50% MeOH extracts ofVerbena officinalis L. (Verbenaceae) leaves. The method enables separation of the main constituents: iridoids, flavonoids and phenolic acid derivatives. Simultaneous detection at different wavelengths, measurement of the UV spectrum of each separated compound during elution and co-injection of reference substances facilitated easy and rapid identification of verbenalin, hastatoside and verbascoside. As some of these compounds, mainly flavonoids, have closely related structures, however, characterization by derivatization with reagents inducing a shift of UV absorption maxima was required. This furnished additional structural information. The reagents were adapted for compatibility with the solvent system used for the chromatographic separation.     

Simultaneous determination of seven phthalic acid esters in beverages using ultrasound and vortex‐assisted dispersive liquid–liquid microextraction followed by high‐performance liquid chromatography

A sensitive, rapid, and simple high‐performance liquid chromatography with UV detection method was developed for the simultaneous determination of seven phthalic acid esters (dimethyl phthalate, dipropyl phthalate, di‐n‐butyl phthalate, benzyl butyl phthalate, dicyclohexyl phthalate, di‐(2‐ethylhexyl) phthalate, and di‐n‐octyl phthalate) in several kinds of beverage samples. Ultrasound and vortex‐assisted dispersive liquid–liquid microextraction method was used. The separation was performed using an Intersil ODS‐3 column (C₁₈, 250 × 4.6 mm, 5.0 μm) and a gradient elution with a mobile phase consisting of MeOH/ACN (50:50) and 0.2 M KH₂PO₄ buffer. Analytes were detected by a UV detector at 230 nm. The developed method was validated in terms of linearity, limit of detection, limit of quantification, repeatability, accuracy, and recovery. Calibration equations and correlation coefficients (> 0.99) were calculated by least squares method with weighting factor. The limit of detection and quantification were in the range of 0.019–0.208 and 0.072–0.483 μg/L. The repeatability and intermediate precision were determined in terms of relative standard deviation to be within 0.03–3.93 and 0.02–4.74%, respectively. The accuracy was found to be in the range of –14.55 to 15.57% in terms of relative error. Seventeen different beverage samples in plastic bottles were successfully analyzed, and ten of them were found to be contaminated by different phthalic acid esters.     

Enantioseparation of racemic paroxol on an amylose‐based chiral stationary phase by supercritical fluid chromatography

The separation of racemic paroxol, a key precursor of trans‐(?)‐paroxetine, on Chiralpak AD‐H, an amylose‐based chiral stationary phase, by supercritical fluid chromatography was studied. Pulse experiments were investigated using supercritical carbon dioxide modified with methanol (MeOH), ethanol and 2‐propanol at 35°C and 15 MPa. Retention and separation factors were determined under analytical conditions for different mobile phase compositions. Among the modifiers used, MeOH was shown to be the best additive, and 5% v/v of MeOH was the preferable concentration at which selectivity of 1.14 and resolution of 3.0 was obtained. In order to evaluate the potential with respect to preparative separations, the adsorption isotherms of individual enantiomers of paroxol were estimated using the elution by characteristic point method. Isotherm parameters were determined from the overloaded elution profiles that were collected at pressure ranging from 15 to 24 MPa. The isotherms obtained were further validated by comparing experimentally recorded elution profiles with the predictions based on the equilibrium‐dispersive model. The results are important to the process design and optimization of preparative supercritical fluid chromatography application.     

Single‐run HPLC/ESI‐LITMS profiling of ginsenosides in plant extracts and ginseng based products

A rapid single‐run analytical approach suitable to achieve a comprehensive characterization of ginsenosides – the main bioactive compounds present in plant materials from Panax species and ginseng‐based products – was developed. The method is based on high‐performance liquid chromatography coupled with electrospray positive ionization linear ion trap mass spectrometry (HPLC/ESI‐LITMS). The main ions in the ESI‐LITMS spectra were attributed to molecular adducts with sodium and potassium and fragments corresponding to cleavage of the glycosidic bonds. The simplicity of the approach allows laborious sample preparation and sophisticated spectral information‐dependent acquisition to be avoided, and provides an opportunity for rapid screening. The method may replace existing HPLC‐DAD profiling approaches. The results of this study indicate that HPLC/ESI‐LITMS is applicable for quality control purposes on processed products and allows the rapid and direct identification of ginsenosides in crude plant extracts. Copyright © 2014 John Wiley & Sons, Ltd.     

High performance liquid chromatography/electrospray mass spectrometry of Hypericum perforatum extracts

Hypericum perforatum L. (St. John's Wort) is a widely distributed herbaceous perennial plant which has been well known as a medicinal plant since antiquity. In recent years, H. perforatum has received increasing attention for the treatment of depression and other neuralgic disorders. The main constituents of H. perforatum extract include flavonoids, naphthodianthrones, phloroglucinols, essential oils and xanthones. The present work reports the analysis of naphthodianthrones and phloroglucinols in H. perforatum extracts by means of high performance liquid chromatography (HPLC) coupled simultaneously to a diode array detector (DAD) and electrospray mass spectrometry (ESI-MS). Hypericin, pseudohypericin, hyperforin and adhyperforin were separated and identified on the base of their on-line UV and mass spectra. Quantitative analysis of hypericin derivatives in different extracts of H. perforatum using DAD and MS detectors was performed. In addition, direct infusion ESI-MS of H. perforatum extracts was applied to obtain rapid mass fingerprints of constituents present in the sample.     

Simple and rapid screening procedure for pesticides in water using SPE and HPLC/DAD detection

An HPLC method, using a photodiode array detector (DAD) has been developed for the simultaneous screening of pesticides. A solid phase extraction system (SPE) has been combined, off-line, with the HPLC/DAD to isolate, recover and quantify pesticides from water samples at ppb levels. The pesticides are eluted from a Hypersil C₁₈ column 5 μm applying a solvent elution programme with two steps, isocratic and gradient mode, in reverse phase. Full UV spectra from 200 to 400 nm are recorded on-line during the analysis and may be compared to stored spectra. The method has been applied to the determination of pesticides in real water samples.     

Enantioseparation of (RS)‐fexofenadine and enhanced detection as the diastereomeric amide and anhydride derivatives using liquid chromatography‐mass spectrometry

Enantioselective analysis of (RS)‐fexofenadine was carried out by achiral HPLC via a derivatization approach using N‐hydroxy‐benzotriazolyl‐(S)‐naproxen ester (synthesized for this purpose) and three chirally pure amines as chiral derivatizing reagents. There occurred formation of amide and anhydride types of diastereomeric derivatives. These were separated and isolated by HPLC (analytical and preparative). The structures and configurations were verified via recording full‐scan product ion mass spectra using LC‐MS, ¹HNMR spectra, Chem3D Pro 12.0 software and the software Gaussian 09 Rev.A.02 program and hybrid density functional B3LYP with 6‐31G basis set supplemented with polarimetry. Experimental conditions for synthesis and separations were optimized and the elution order was established. Analytical separation was performed on a C₁₈ analytical column with different ratios of MeCN–TEAP buffer and MeOH–TEAP buffer (v/v) adjusted to pH 7.5 as mobile phase at a flow rate of 0.7 mL min^‐1. Detection was performed via UV absorbance at 225 nm. The method was validated in accordance with International Conference on Harmonization guidelines. The detection limits were 6.25 and 7.87 ng mL^‐1 for first and second eluting diastereomeric derivatives, respectively.     

Analytical and semi‐preparative enantioresolution of (RS)‐ketorolac from pharmaceutical formulation and in human plasma by HPLC

An efficient, simple, validated, analytical and semi‐preparative HPLC method has been developed for direct enantioresolution of (RS)‐Ketorolac (Ket) using monochloro‐methylated derivatives of cellulose and amylose, i.e. cellulose (tris‐3‐chloro‐4‐methylphenylcarbamate) and amylose (tris‐5‐chloro‐2‐methylphenylcarbamate) as chiral stationary phases (CSPs) with photo diode array detection at 320 nm. Enantioresolution was carried out in samples of human plasma spiked with (RS)‐Ket under normal and reversed‐phase elution modes with suitable mobile phase compositions. The effect of nature of alcohols (MeOH, EtOH, PrOH and n‐BuOH) and other solvents (MeCN and MeOH) as organic modifiers in the mobile phase was investigated on the separation performance of two CSPs in terms of retention and separation of enantiomers. The best resolution was observed on cellulose‐based CSP using EtOH, while using 2‐PrOH (15%) and amylose‐based CSP obtained the highest retention. Under reversed‐phase elution mode the best enantioseparation was observed using 30% MeCN with ammonium formate buffer. The elution order of enantiomers was ascertained by determining specific rotations. The limit of detection and quantitation values were 5 and 15.5 ng/mL for each enantiomer of (RS)‐Ket, respectively. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a high‐performance liquid chromatography method for the quantification of talazoparib in rat plasma: Application to plasma protein binding studies

A sensitive and selective RP‐HPLC method has been developed and validated for the quantification of a highly potent poly ADP ribose polymerase inhibitor talazoparib (TZP) in rat plasma. Chromatographic separation was performed with isocratic elution method. Absorbance for TZP was measured with a UV detector (SPD‐20A UV–vis) at a λ_max of 227 nm. Protein precipitation was used to extract the drug from plasma samples using methanol–acetonitrile (65:35) as the precipitating solvent. The method proved to be sensitive and reproducible over a 100–2000 ng/mL linearity range with a lower limit of quantification (LLQC) of 100 ng/mL. TZP recovery was found to be >85%. Following analytical method development and validation, it was successfully employed to determine the plasma protein binding of TZP. TZP has a high level of protein binding in rat plasma (95.76 ± 0.38%) as determined by dialysis method.     

Versatile chromatographic method for catechin determination in development of topical formulations containing natural extracts

Catechin is found in several natural sources, as Eugenia dysenterica and Syzygium cumini extracts. Its antioxidant and UV‐protective properties suggest a potential use in cosmetic and dermatological formulations. A simple analytical method capable of giving support to experiments performed along the development of topical formulations containing this natural substance (i.e. drug assay, skin permeation and stability studies), however, is still needed. Thus, this work aimed to develop and validate a selective HPLC method for catechin determination during the development of topical formulations. Separation was achieved using an RP‐C₁₈ column (300 × 3.9 mm; 10 μm), with a mobile phase of methanol–phosphoric acid 0.01 m (15: 85, v/v), a flow rate of 0.8 mL/min, temperature set at 40°C and UV detection at 230 nm. The method was linear in a range from 0.5 to 10.0 μg/mL (r = 0.9998), precise with an overall variation coefficient of 5.5% and accurate with catechin recovery from the skin layers >85%. Additionally, the method was sensitive (limit of detection, 0.109 μg/mL; limit of quantification, 0.342 μg/mL) and selective against plant extracts, skin matrices and formulation interferents, as well as catechin degradation products. It was also robust regarding both methodology parameters and analytical stability.     

Quantitative analysis and chromatographic fingerprinting of the semen zizyphi spinosae by ultra-high-performance liquid chromatography coupled with diode-array detector

A simple and sensitive method was developed and validated for fingerprint analysis of semen zizyphi spinosae (SZS) and simultaneous determination of six flavonoids in SZS by ultra‐high‐performance liquid chromatography coupled to diode‐array detector (DAD). The analysis was performed on an Agilent ZORBAX Eclipse Plus C18 RRHD column. The column was maintained at 40°C and the eluents were monitored with DAD at 270 nm. A gradient elution of acetonitrile and water containing 20 mM sodium dihydrogen phosphate was used. The solvent flow rate was 0.4 mL/min. The method was validated. Standard calibration curves showed good linear behaviors (r=1.000) in the range of 0.33–201.00 μg/mL. Acceptable intra‐day precision (RSD<1.9%), inter‐day precision (RSD<4.0%), repeatability (RSD<4.1%) and recovery in the range of 97.4–104% were obtained. The validated method was successfully applied to obtain the chromatographic fingerprints and the contents of six flavonoids in 23 samples of SZS. The principal component analysis (PCA) had been applied for the chromatographic fingerprint analysis and quantitative analysis of six flavonoids to classify and discriminate the 23 samples of SZS. These results demonstrated that the method was very suitable in the analysis and quality control of SZS.