High Performance Liquid Chromatography Coupled To Diode Array Detection Research Articles

DETERMINATION OF TRICLABENDAZOLE IN CATTLE PLASMA AS ITS SULPHOXIDE AND SULPHONE METABOLITES BY ROTATING DISK SORPTIVE EXTRACTION COMBINED WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND ITS APPLICATION TO PHARMACOKINETIC STUDIES

In this study, a new method for determination of triclabendazole (TCB) as its main metabolites, triclabendazole sulfoxide (TCB-SO) and triclabendazole sulfone (TCB-SO2) in animal plasma was developed. TCB is widely used as antiparasitic in the veterinary industry. Rotating disk sorptive extraction (RDSE) was the selected sample preparation technique for extraction/clean up of the compounds from the samples followed by high performance liquid chromatography coupled to diode array detection (HPLC-DAD) for quantification. Optimization of physicochemical variables was performed using a multivariate experimental design. Following the recommendations given in the Veterinary International Conference on Harmonization guides (VICH GL02 and VICH GL49), the validation of the method was performed, given rise to improved analytical features compared with those provided by other methods based on solid phase extraction (SPE). Linearity (r > 0.99) was achieved for both compounds when calibration was performed not only in standard solutions but also in animal spiked plasma. Selectivity, defined as the response ratio between blank and analyte at the limit of quantification, was 11.8% and 3.4% for TCB-SO and TCB-SO2, respectively. Accuracy and precision, expressed in percentage, were always lower than -16.7% and 8.1%, respectively. Eco-efficiency was quantitatively assessed indicating that the method is an excellent green method. The proposed analytical method was applied to the determination of the pharmacokinetic of two commercial products of TCB in cattle plasma after oral administration. The good analytical performance, eco-efficiency and economy, make the method interesting for alternative use in routine laboratories.

Strategies to Document Adulteration of Food Supplement Based on Sea Buckthorn Oil: a Case Study

In this case study, a food supplement with declared content of sea buckthorn oil was investigated since it was suspected to be adulterated by sunflower oil. Polar and non-polar fractions extracted from oil supplement were analyzed and compared with authentic sea buckthorn and sunflower oils examined in the same way. Three different analytical platforms were used in order to characterize these samples: (i) ambient mass spectrometry consisting of direct analysis in real time (DART) ion source coupled to quadrupole time-of-flight mass spectrometry (Q-TOF MS); (ii) ultra performance liquid chromatography (UHPLC) coupled to quadrupole time-of-flight mass spectrometry, and (iii) high-performance liquid chromatography coupled to diode array detector (HPLC-DAD). The fingerprints of polar and non-polar extracts, regardless they were obtained by DART-HRMS and/or UHPLC-HRMS, were almost identical with those of sunflower “reference” oil. The last of employed techniques, HPLC-DAD, provided complementary information on the occurrence of various visible light-absorbing compounds. While a rich carotenoid profile was shown in sea buckthorn oil, dominated by β-carotene, lutein, and lycopene, the color of the suspected sample was caused only by high concentration of β-carotene. In principle, these techniques proved to be suitable and complementary tools for oil authentication and allowed an oil supplement to be rapidly verified.

European marketable grain legume seeds: Further insight into phenolic compounds profiles

Twenty-nine mature raw varieties of grain legume seeds (chickpeas, field peas, faba beans, common vetch and lupins) produced in Europe were investigated for their phenolic profile by means of high performance liquid chromatography coupled to diode array detection (HPLC-DAD). To the best of our knowledge, this study reported for the first time the phenolic composition of mature raw seeds of chickpea type Desi, field pea and common vetch. Phenolic acids were predominant compounds in chickpeas, field peas and common vetch compared to flavonoids, whereas the opposite was observed for lupin seeds. Yellow lupins presented the highest levels of total phenolic compounds followed by narrow-leafed lupins (in average 960 and 679mg/kg, dry basis, respectively), whereas Kabuli chickpeas got the lowest ones (in average 47mg/kg, dry basis). Principal component analysis revealed that flavones and total levels of phenolic compounds were responsible for nearly 51% of total data variability.

A Rapid and Robust HPLC- DAD Method for the Monitoring of Thiopurine Metabolites in Whole Blood: Application to Paediatric Patients with Inflammatory Bowel Disease

Mercaptopurine (MP) and azathioprine (AZA) are thiopurine drugs that are used in paediatrics for the treatment of inflammatory bowel disease (IBD), Therapeutic drug monitoring (TDM) of thiopurine active metabolites, 6-methylmercaptopurine (6MMP) and 6-thioguanine nucleotides (6-TGN) is a common practice for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new High Performance Liquid Chromatography coupled to Diode Array Detection (HPLC-DAD) method for the simultaneous quantification of 6-TGN and 6-MMP in whole blood with a total runtime of 7.5 minutes over a wide range of concentrations (50 – 2000 pmol/8x10^8 RBC for 6-TG and 20 – 15000 pmol/8x10^8 RBC for 6-MMP respectively). The new method has been validated following international guidelines on bioanalytical method validation. Within- and between-run precision and accuracy results for quality control samples were always ≤ 15% of the nominal concentrations. The correlation with our reference method was excellent. The new HPLCDAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses.

Rapid determination of baicalin and total baicalein content in Scutellariae radix by ATR-IR and NIR spectroscopy

In this study methods for the quantification of baicalin and total baicalein in Scutellariae radix with near infrared (NIR) spectroscopy and attenuated-total-reflectance mid-infrared (ATR-IR) spectroscopy in hyphenation with multivariate analysis were developed and compared. The reference analysis was performed by high performance liquid chromatography coupled to diode array detection (HPLC-DAD). Different pretreatments like standard normal variate (SNV), multiplicative scatter correction (MSC), first and second derivative Savitzky-Golay were applied on the spectra to optimize the calibrations. A principal component analysis was performed with both spectroscopic methods to distinguish wild and cultivated samples. Quality parameters obtained for test-set calibration models of ATR-IR spectroscopy (baicalin: standard error of prediction (SEP)=1.31, ratio performance to deviation (RPD)=2.91 and R2=0.88; total baicalein: SEP=1.02, RPD=3.24 and R2=0.89) and NIR spectroscopy (baicalin: SEP=1.50, RPD=2.54 and R2=0.88; total baicalein: SEP=1.19, RPD=2.76 and R2=0.84) demonstrate that both spectroscopic techniques in combination with multivariate analysis are successful tools for the quantification of baicalin and total baicalein in Scutellariae radix, but it was found that ATR-IR spectroscopy provides higher accuracy in the given application. Furthermore it was proved that wild and cultivated samples can be distinguished by ATR-IR.

Development of a simple analytical method for the simultaneous determination of paracetamol, paracetamol-glucuronide and p-aminophenol in river water

Paracetamol is among the most worldwide consumed pharmaceuticals. Although its occurrence in the environment is well documented, data about the presence of its metabolites and transformation products is very scarce. The present work describes the development of an analytical method for the simultaneous determination of paracetamol, its principal metabolite (paracetamol-glucuronide) and its main transformation product (p-aminophenol) based on solid phase extraction (SPE) and high performance liquid chromatography coupled to diode array detection (HPLC-DAD). The method was applied to analysis of river waters, showing to be suitable to be used in routine analysis. Different SPE sorbents were compared and the use of two Oasis WAX cartridges in tandem proved to be the most adequate approach for sample clean up and pre-concentration. Under optimized conditions, limits of detection in the range 40-67ng/L were obtained, as well as mean recoveries between 60 and 110% with relative standard deviations (RSD) below 6%. Finally, the developed SPE-HPLC/DAD method was successfully applied to the analysis of the selected compounds in samples from seven rivers located in the north of Portugal. Nevertheless all the compounds were detected, it was the first time that paracetamol-glucuronide was found in river water at concentrations up to 3.57μg/L.

Pressurized Hot Water Extraction of Alkaloids in Goldenseal

A quick, efficient and environmentally beneficial approach for the extraction of alkaloids in goldenseal based on pressurized hot water extraction (PHWE) is presented. PHWE was evaluated following the extraction of hydrastine and berberine which are the two main alkaloids of goldenseal. The effects of temperature and other parameters on extraction yields were monitored by high performance liquid chromatography coupled to diode array detector (HPLC-DAD). The extraction yields were compared to those achieved through reflux and ultrasonic extraction methods. PHWE employed hot water at an optimal temperature of 140℃, a pressure of 50 bars and flow rate of 1 mL.min-1 for extraction experiments conducted in the dynamic mode. Both PHWE and conventional methods achieved comparable extraction yields. Reflux and ultrasonic extractions had procedures that were slower (over 6 h) and employed large quantities (200 mL) of organic solvents. The PHWE method was simple and relatively fast as extraction was achieved in 15 min. At 95% confidence level (n = 3), reflux and ultrasonic methods recorded extraction yields that were not significantly different from that of PHWE. Moreover, PHWE employed a more readily available “green” solvent, (water) to achieve comparable results to those of conventional methods which utilized organic solvents. The percentage relative standard deviations (%RSD) for the extraction yields were less than 3% in all cases indicating the high reproducibility of the method. However, when using PHWE to extract thermally labile analytes like alkaloids, degradation effects also occurred. Increasing the temperatures certainly achieved higher extraction yields but also led to degradation of these thermally labile compounds indicating a necessity for optimization.

Preparation and evaluation of antioxidant capacity of Jackfruit ( Artocarpus heterophyllus Lam.) wine and its protective role against radiation induced DNA damage

Jackfruit is an underutilized edible fruit in the tropics and subtropics. The aim of this study was to produce wine from jackfruit pulp and to evaluate the total phenolic and flavonoid contents and antioxidant properties of the wine. The ability of scavenging free radicals was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant assay (FRAP), N, N-dimethyl-p-phenylendiamine (DMPD) and nitric oxide (NO) scavenging assays. Experimental results indicated that jackfruit wine was effective in DPPH radical scavenging (69.44 ± 0.34%), FRAP (0.358 optical density value, O.D.), DMPD (78.45 ± 0.05%) and NO (62.46 ± 0.45%) capacity. By the analysis of the high performance liquid chromatography coupled to diode array detector (HPLC-DAD), two phenolic compounds namely gallic acid and protocatechuic acid were identified. The jackfruit wine was also able to protect H 2O 2 + UV radiation and γ-radiation (100 Gy) induced DNA damage in pBR322 plasmid DNA. The antioxidant and DNA damage protecting properties of jackfruit wine confirmed health benefits when consumed and could become a valuable source of antioxidant rich neutraceuticals. Additionally, the wine could be a commercially valuable by-product for the jackfruit growers.

Application of molecularly imprinted polymer solid-phase extraction for salivary cotinine

A method constituted by molecularly imprinted solid-phase extraction (MISPE) with high-performance liquid chromatography coupled to diode array detector (HPLC-DAD) was developed for cotinine analysis in saliva samples. For this purpose, the separation was carried out with a C18 reversed-phase column at 20 °C. The mobile phase which was composed of a mixture of 09:91 (v/v) acetonitrile/phosphate buffer, pH 6.3, was delivered with isocratic flow rate at 1.4 mL min(-1). Employing MISPE, the best conditions were achieved with 1.5 mL of saliva plus 1.5 mL of 0.1 mol L(-1) of acetate buffer, pH 5.5, which were then passed through a cartridge previously conditioned with 2 mL acetonitrile, 2 mL methanol, and 2 mL of 0.1 mol L(-1) sodium acetate buffer, pH 5.5. The washing was carried out with 1 mL deionized water, 1 mL of 0.1 mol L(-1) sodium hydroxide, and 1 mL hexane; finally; the cotinine elution was carried out with 3 mL methanol/water (97.5: 2.5, v/v). Linearity ranged from 30 to 500 ng mL(-1) with r > 0.99. Intra-assay, interassay precision, and accuracy ranged from 3.1% to 10.1%, 5.2% to 15.9%, and 99.22% to 111.17%, respectively. The detection and quantification limits were 10 and 30 ng mL(-1), respectively. This investigation has provided a reliable method for routine cotinine determination in saliva, and it is an important tool for monitoring cigarette smoke exposure in smokers. The method was applied in five smokers' samples who consumed around five to 20 cigarettes per day and the values of cotinine in saliva were from 66.7 to 316.16 ng mL(-1).

Further Insight into the Latex Metabolite Profile of Ficus carica

Latex is a sticky emulsion that exudes upon damage from specialized canals from several plants. It contains several biologically active compounds, such as phytosterols, fatty acids, and amino acids. In plants, these compounds are involved in the interaction between plants, insects, and the environment. Despite its chemical, biological, and ecological importance, Ficus carica latex is still poorly studied. To improve the knowledge on the metabolite profile of this matrix, a targeted metabolite analysis was performed in a representative sample from F. carica latex. Seven phytosterols were determined by gas chromatography-ion trap mass spectrometry (GC-ITMS) and high-performance liquid chromatography coupled to diode array detection (HPLC-DAD), with β-sitosterol and lupeol being the compounds present in higher concentrations (ca. 54 and 14%, respectively). A total of 18 fatty acids were characterized by GC-ITMS, being essentially represented by saturated fatty acids (ca. 86.4% of total fatty acids). A total of 13 free amino acids were also identified by high-performance liquid chromatography coupled to ultraviolet-visible spectroscopy (HPLC/UV-vis), and cysteine and tyrosine were the major ones (ca. 38.7 and 31.4%, respectively). In humans, phytosterols and some polyunsaturated fatty acids, such as linoleic acid, are known for their anticarcinogenic properties. With regard to amino acids, some of them, such as glycine, are neurotransmitters. Our results reveal the presence of a wide diversity of compounds, from distinct classes, in F. carica latex, possessing various potential pharmacological activities; thus, its biological potential appears to be worth further exploring.

Passiflora incarnata L. (Passionflower) extracts elicit GABA currents in hippocampal neurons in vitro, and show anxiogenic and anticonvulsant effects in vivo, varying with extraction method

Potential mechanisms of Passiflora incarnata extracts and the effect of extraction methods on ingredients and biological effects were explored. Using the same batch of plant material, total flavonoid yields as measured by high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) increased substantially with hot versus cold extraction methods. Whole Passiflora extract induced prominent, dose-dependent direct GABA(A) currents in hippocampal slices, but the expected modulation of synaptic GABA(A) currents was not seen. GABA was found to be a prominent ingredient of Passiflora extract, and GABA currents were absent when amino acids were removed from the extract. Five different extracts, prepared from a single batch of Passiflora incarnata, were administered to CF-1 mice for 1 week in their drinking water prior to evaluation of their behavioral effects. Anticonvulsant effects against PTZ-induced seizures were seen in mice that received 2 of the 5 Passiflora extracts. Instead of the anxiolytic effects described by others, anxiogenic effects in the elevated plus maze were seen in mice receiving any of the 5 Passiflora extracts.

Flavonoid contents and antioxidant activities from Cinnamomum species

Cinnamomum has long been regarded as a food or medicinal plant. Leaves of five species of Cinnamomum, namely C. burmanni, C. cassia, C. pauciflorum, C. tamala and C. zeylanica, were chosen to investigate their antioxidant activities in this study. C. zeylanica exhibited the highest total phenolic content while C. burmanni had the highest flavonoid content among the five species. These five species were then screened for their antioxidant potentials using various in-vitro models such as total antioxidant capability, 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity, reducing power and superoxide anion scavenging activity at various concentrations. C. zeylanica showed the highest DPPH radical scavenging activity, total antioxidant activity and reducing power, while C. tamala exhibited the highest superoxide anion scavenging activity. By the analysis of the high performance liquid chromatography coupled to diode array detector (HPLC-DAD), three flavonoid compounds namely quercetin, kaempferol and quercetrin were identified and quantified. This study suggested that Cinnamomum leaf can be used potentially as a readily accessible source of natural antioxidants. This study was focused to evaluate the antioxidant activities of five species of Cinnamomum leaf which is normally used in medicine and also used in food preparation. This study provided an alternative of utilizing Cinnamomum leaf as a readily accessible source of natural antioxidants in food and pharmaceutical industry.

Matrix dependent impact of sugar and ascorbic acid addition on color and anthocyanin stability of black carrot, elderberry and strawberry single strength and from concentrate juices upon thermal treatment

Abstract Heat stability of strawberry, elderberry and black carrot juices prepared from concentrate and fresh plant material was investigated at pH 3.5 by heating at 95 °C for 2 and 4 h, respectively. The impact of added saccharides (glucose, fructose, sucrose) and ascorbic acid on 5-hydroxymethylfurfural (HMF) formation, browning together with the stability of anthocyanins and their color properties were assessed using high performance liquid chromatography coupled to diode array detection (HPLC-DAD) and ultraviolet–visible (UV–Vis) spectrophotometry. Thermal degradation products of anthocyanins and saccharides were identified using liquid chromatography coupled to multistage mass spectrometry (LC–MSn). Pigment stability and color changes depended on anthocyanin structure and juice matrix. Whereas a slight but insignificant stabilizing effect of added sugars and ascorbic acid was observed in juice prepared from black carrot concentrate, obvious color differences were detected in strawberry and elderberry juices when ascorbic acid was added. Manually squeezed juices from fresh plant material showed higher color stability compared to juices prepared from concentrate which might be due to the retention of polymeric matrix compounds in the former. The data presented may contribute to the development of advanced technological processes for the production of coloring foodstuff and food with improved color stability.

Quantification of 3 R,3′ R-zeaxanthin in plant derived food by a diastereomeric dilution assay applying chiral high-performance liquid chromatography

For quantification of 3 R,3′ R-zeaxanthin in plant derived food, a new diastereomeric dilution assay (DIDA) was developed using chiral high-performance liquid chromatography coupled to diode-array detection (HPLC/DAD). As diastereomeric internal standard (I.S.), 3 R,3′ S-( meso)-zeaxanthin was applied. DIDA showed sufficient sensitivity with low limits of detection and quantification (18 μg 100 g −1 and 45 μg 100 g −1), a good inter-assay coefficient of variation (2.4%), determined by analysis of a certified BCR reference material, and a good recovery (94.7%). To prove the applicability of DIDA, various plant materials were anaylzed, showing that DIDA is highly advisable if saponification is applied during work-up to hydrolyze xanthophyll esters.

A perennial bloom of Planktothrix agardhii (Cyanobacteria) in a shallow eutrophic French lake: limnological and microcystin production studies

A perennial bloom of the hepatotoxic filamentous cyanobacterium Planktothrix agardhii was investigated from January 1999 to February 2000 in a shallow eutrophic lake in the south of Paris (Viry-Châtillon, France). The lake is linked to the Seine River by an artificial channel and used for both recreation and fishing. Little data on cyanobacterial blooms is available in France. The aim of this preliminary study was to characterise the cyanobacterial bloom limnologically as well as for toxicity levels at one site in the lake and another in the channel. A relatively high biomass of P. agardhii was maintained throughout the year with maximum density in winter (7.9 ×10 4 to 4.5×10 6 trichomes/L). The only other significant phytoplankton species in the water bodies was also a cyanobacterium: Limnothrix redekei. The nutrient levels in the lake, although still relatively high (SRP varied in the range between 0.13 and 2 μM but NO 3 - stayed under the detection limit), were generally lower than in the channel which reflected the hydraulic influence of exchanges with the eutrophic Seine River. The concentration of microcystins (hepatotoxic cyclic heptapeptides) was measured in bloom extracts throughout the year using two different techniques: protein phosphatase 2A inhibition assay and high performance liquid chromatography coupled to diode array detection (HPLC-DAD). High microcystin concentrations, up to 5.2 μg/L MCYST-LR eq., were found at the two sampling sites, with good correlation between the two methods. The presence of five different microcystins was confirmed by further analysis of samples by LC-MS. Factors affecting seasonal population bloom dynamics, toxicity and the possible public health significance of these toxic cyanobacterial populations in the lake are discussed.

Antibiotic residues in milk

A novel application of rotating disk sorptive extraction (RDSE), using a styrene-divinylbenzene (S-DVB) sorbent for the extraction of antibiotic residues in milk was developed. The analytes studied were oxytetracycline and its 4-epimer, enrofloxacin, ciprofloxacin, sulfadoxine and trimethoprim. After RDSE, the analytes were determined by performing both high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) and ultrahigh-performance liquid chromatography coupled to ultraspray-electrospray-time of flight-mass spectrometry (UPLC-TOF/MS).By using HPLC-DAD, the absolute recoveries were between 85.5% and 106.4% with relative standard deviations between 3.7% and 9.9%. The obtained limits of quantification (LOQs) were lower than the respective maximum residue levels (MRLs) reported for each analyte, demonstrating that the methodology was applicable for residue depletion studies. UPLC-TOF/MS showed absolute recoveries from 88.5% to 114.1%, with RSDs between 4.3% and 15.4%. The LOQs obtained using UPLC-TOF/MS were also lower than the MRLs for each respective analyte.Compared with other analytical methods previously reported for some of the analytes, the present method is simpler and less expensive and utilizes green chemistry, all while providing comparable figures of merit.