High-performance Liquid Chromatographic Determination Research Articles

Modified vortex-assisted dispersive liquid–liquid microextraction coupled to high-performance liquid chromatography for determination of amino acids in human plasma and seawater specimens

Modified vortex-assisted dispersive liquid–liquid microextraction coupled to high-performance liquid chromatography for determination of amino acids in human plasma and seawater specimens

Solid-liquid-solid conversion microextraction combined with high-performance liquid chromatography for determination of bisphenols

A novel solid-liquid-solid conversion microextraction combined with high-performance liquid chromatography method (SLSC-ME-HPLC-UV) has been developed to preconcentrate trace bisphenols (BPs) in environmental water, in which poly (isopropyl acrylamide-co-methyl methacrylate) P(MMA-co-NIPAM) was used as the extractant. The hydrogen bond and hydrophobic interaction play an important role between the P(MMA-co-NIPAM) and BPs. The ratio of copolymer MMA to NIPAM monomers, type and concentration of salt, extraction time and acetonitrile volume, as variables affecting the efficiency of extraction process, were investigated with the aid of single-variable optimization. Under optimal conditions, the coefficient of determination (R2) of calibration curves was greater than 0.999. Enrichment factors (EFs) ranged from 74 to 128. Extraction recoveries (ERs) ranged from 44 to 77%. The limits of detection (LODs) and limits of quantification (LOQs) were in the ranges of 0.20–0.90 and 0.68–3.01 μg L−1, respectively. The values for intra-day and inter-day precisions were lower than 3.6% and 3.3%, respectively.

Preparation of new hydrophobic deep eutectic solvents and their application in dispersive liquid–liquid microextraction of Sudan dyes from food samples

In this work, a new generation of hydrophobic deep eutectic solvents (DESs) was prepared using eugenol (as hydrogen bond donor) and benzyltriethylammonium bromide, benzyltributylammonium bromide, benzyltriethylammonium chloride and benzyltributylammonium chloride (as hydrogen bond acceptor) in different molar ratios. These DESs were applied to vortex-assisted dispersive liquid-liquid microextraction of Sudan dyes from food samples, followed by high-performance liquid chromatographic determination. The influencing parameters, including the type of DES, amount of DES, extraction time, solution pH and salt addition, were investigated and optimized. Under the optimized conditions, a linear range of 2-1000ngmL-1 with determination coefficients of <0.999 was obtained. Limits of detection and limits of quantification were in the range of 0.5 to 1ngmL-1 and 2 to 3ngmL-1, respectively. The proposed method was successfully used in the determination of Sudan dyes in chili sauce, chili powder and ketchup, and satisfactory recoveries of between 89.9 and 119.3% were obtained, with relative standard deviations in the range of 0.1-6.8%. The proposed method is simple, green and efficient, and can be applied to determine Sudan dyes in complex matrices.

Synthesis of value-added MIL-53(Cr) from waste polyethylene terephthalate bottles for the high-performance liquid chromatographic determination of methylxanthines in tea

In this work, which was based on the concepts of green chemistry, polyethylene terephthalate (PET) waste bottles were employed as the source of a benzene-1,4-dicarboxylate linker for the synthesis of MIL-53(Cr). Subsequently, the synthesized MIL-53(Cr) was characterized and employed for the first time as a stationary phase for the HPLC determinations of different methylxanthines in black, green, and white tea samples, respectively. As observed, the main working mechanism was the reversed-phase; however, size-exclusion effects were noticeable on catechins, which exceeded the dynamic diameter of MIL-53(Cr) (~8.5 Å). Under optimal conditions, the MIL-53(Cr) column exhibited the best efficiency for caffeine with 35,300 plates m−1 and a chromatographic resolution of ≥1.72 for all the detected compounds in the tea samples. The developed method was linear (0.183–200 μg mL−1) and the obtained LOD values were 0.055–0.072 μg mL−1. The obtained RSDs, which were determined employing different chromatographic parameters indicated the feasible reproducibility of the columns and verified the ruggedness of the method. The results confirmed that the methylxanthine contents of the black teas were the highest (51.28–60.02 mg per tea bag; 2.0 g), followed by those of the green teas (26.72–43.68 mg per tea bag), before those of the white teas (20.44–25.36 mg per tea bag). Furthermore, the separation performances of the MIL-53(Cr) column and two silica-based C18 commercially columns (3 and 5 μm-sized particles) were compared. The structure of the MIL-53(Cr) afforded higher retention factors in the methylxanthines because of the hydrophobic π–π interactions between the solutes and aromatic ring of the organic linker. Conclusively, the MIL-53(Cr) column, which was prepared from PET bottles, could be applied to the routine analyses of methylxanthines in tea samples.

HPLC Determination of Formaldehyde in Flour Samples using 2,4,6-Trichlorophenyl Hydrazine as Derivatization Reagent

A new simple and sensitive high-performance liquid chromatography (HPLC) method for the determination of formaldehyde in flour samples has been developed. Formaldehyde was quantified after derivatization with a readily available reagent, 2,4,6-trichlorophenyl hydrazine (TCPH) under basic conditions. The formaldehyde-TCPH derivative was eluted with chromatographic mobile phase of 70:30 (v/v) acetonitrile:water at a flow rate of 1.0 mL min–1; wavelength, 222 nm; injection volume, 50 μL, using a C18 ODS Hypersil column (250 mm × 4.5 mm, 5 μm). The calibration curve was linear over the range of 0.001-10 μg mL-1 with R2 = 0.999. Recoveries at three different concentration levels (0.1, 1.0 and 5 μg mL-1) ranged from 92.0-101.7% with RSD less than of 2.2%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.3 and 1.0 ng mL-1, respectively. The developed method was used for the determination of formaldehyde in various flour-based samples.

High performance liquid chromatography determination of L-glutamate, L-glutamine and glycine content in brain, cerebrospinal fluid and blood serum of patients affected by Alzheimer's disease.

Altered glutamatergic neurotransmission is thought to play a crucial role in the progression of Alzheimer's disease (AD). Accordingly, the identification of peculiar biochemical patterns reflecting AD-related synaptopathy in blood and cerebrospinal fluid (CSF) could have relevant diagnostic and prognostic implications. In this study, we measured by High-Performance Liquid Chromatography the amount of glutamate, glutamine and glycine in post-mortem brain samples of AD patients, as well as in CSF and blood serum of drug-free subjects encompassing the whole AD clinical spectrum (pre-clinical AD, n = 18, mild cognitive impairment-AD, n = 29, dementia AD, n = 30). Interestingly, we found that glutamate and glycine levels, as well as total tau protein content, were significantly reduced in the superior frontal gyrus of patients with AD, compared with non-demented controls. No significant change was also found in glutamate, glutamine and glycine CSF concentrations between AD patients and neurological controls. Remarkably, serum glutamate levels were significantly higher in patients affected by early AD phases compared to controls, and were negatively correlated with CSF total tau levels. Conversely, serum glutamine concentration was significantly increased in AD patients, with a negative correlation with MMSE performances. Finally, we reported a significant correlation between serumL-glutamate concentrations and CDR score in female but not in male cohort of AD subjects. Overall, our results suggest that serum glutamate and glutamine levels in AD patients could vary across disease stages, potentially reflecting the progressive alteration of glutamatergic signaling during neurodegenerative processes.

Enzymatic determination of carnosine in meat and fish using β-Ala-Xaa dipeptidase and histidine ammonia-lyase derived from Pseudomonas putida NBRC100650.

Carnosine is a naturally occurring dipeptide and a functional component in foods, while also showing health-promoting effects. Generally, food-derived carnosine is quantified via high-performance liquid chromatography (HPLC). We have developed a method for quantifying carnosine in foods using microbial enzymes, β-Ala-Xaa dipeptidase (BapA) and histidine ammonia-lyase (HAL). The carnosine concentrations in extracts of chicken, pork, beef, bonito, and tuna were determined via both HPLC and enzymatic determination. The carnosine contents measured via enzymatic determination were in agreement with those determined via conventional HPLC analysis. Relative standard-deviation values of the conventional HPLC method and the enzymatic determination of carnosine in foods were 0.728-5.76% and 0.504-4.58%, respectively. The recovery of carnosine in food extracts via enzymatic determination was 97-103%. Therefore, the developed enzymatic determination technique using BapA and HAL can be used for the determination of carnosine in meats and fishes with comparable accuracy to that of conventional HPLC analysis.

A novel stability-indicating method for determination of a new antidepressant effect of vortioxetine in a pharmaceutical formulation by using RP-HPLC

BackgroundA novel rapid, accurate, and stability-indicating reversed-phase high performance liquid chromatographic (RP-HPLC) and first derivative spectrophotometric determination were explained for the assay of vortioxetine (VRT) in bulk and pharmaceutical formulations. For RP-HPLC method, optimal separation and determination of VRT were achieved with a Waters Symmetry C18, (100 × 4.6 mm, 3.5 μm) analytical column using a mobile phase consisting of methanol:0.05 M potassium dihydrogen phosphate (pH:3.0 ± 0.05) (30:70, v/v) in isocratic mode with flow rate of 1.3 mL min−1. Injection volume was 20 μL. The maximum absorption wavelength of VRT is 225.0 nm; hence, 225.0 nm was studied as the detection wavelength and column at 50 °C temperature. The caffeine was used as the internal standard (IS). On the other hand, the first derivative spectrophotometric method for the analysis of vortioxetine was performed by measuring the amplitude at 251.7 and 272.6 nm.ResultThe HPLC method was found to be linear in the concentration ranges of 10.0-70.0 μg mL−1 with the coefficient value R2 of 0.9998, and the mean recovery value was 100.7%. Further stability studies were done through exposure of the analyte solutions to various stress conditions: acid, alkali hydrolysis, chemical oxidation, and exposure to UV radiation. For the first derivative spectrophotometric method, linearity was observed in the concentration range 6.0-30.0 μg mL−1 (for 237.7 nm R2 = 0.9999 and for 257.2 nm R2 = 0.9997).ConclusionThe methods were validated in accordance with ICH guidelines with respect to linearity, accuracy, specificity, limit of detection, precision, and limit of quantification.

Deep eutectic solvent-based liquid-phase microextraction coupled with reversed-phase high-performance liquid chromatography for determination of α-, β-, γ-, and δ-tocopherol in edible oils.

For simultaneous analysis of four fat-soluble tocopherols (α-, β-, γ-, and δ-) in edible oils, an efficient and green method using deep eutectic solvent-based liquid-phase microextraction (DES-LPME) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) was developed. The DESs formed by different quaternary ammonium salts and ethanol were used as the extractants. Tetrabutylammonium chloride (TBAC)-ethanol DES at a molar ratio of 1:2 achieved the best extraction efficiency. Under the optimized conditions, the detection limits were in the range of 2.1-3.0ngmL-1. The intra-day and inter-day repeatability were in the ranges of 3.9-5.3% and 4.8-7.1%, respectively, and the recoveries for the real samples varied from 80.7% to 105.4%. The developed method was successfully employed for the determination of all four tocopherol homologues with an RP-HPLC system containing a COSMOSIL π-NAP column in five edible oils collected locally. Graphical abstract.

High performance liquid chromatographic (HPLC) determination of available lysine in milk protein-maize composite extrudates and its stability during storage

High performance liquid chromatographic (HPLC) determination of available lysine in milk protein-maize composite extrudates and its stability during storage

High-Performance Liquid Chromatographic Determination of Chiral Amino Acids Using Pre-Column Derivatization with &lt;i&gt;o&lt;/i&gt;-Phthalaldehyde and &lt;i&gt;N&lt;/i&gt;-&lt;i&gt;tert&lt;/i&gt;-Butyloxycarbonyl-D-cysteine and Application to Vinegar Samples

High-Performance Liquid Chromatographic Determination of Chiral Amino Acids Using Pre-Column Derivatization with &lt;i&gt;o&lt;/i&gt;-Phthalaldehyde and &lt;i&gt;N&lt;/i&gt;-&lt;i&gt;tert&lt;/i&gt;-Butyloxycarbonyl-D-cysteine and Application to Vinegar Samples

Stability-Indicating High-Performance Liquid Chromatographic Determination of Levamisole Hydrochloride in Bulk and Dosage Forms

A selective, accurate, and precise stability-indicating HPLC method for analysis of levamisole hydrochloride in bulk and in their injection and oral solution formulations has been developed and validated in accordance with ICH guidelines. The chromatographic separation was carried out on a C8 column (250 × 4.6 mm with a particle size of 5 micrometer) using a mixture of phosphate buffer pH 8.0 and acetonitrile (70 : 30 v/v) as the mobile phase pumped at a flow rate of 1.5 ml/min with UV detection at 215 nm. The calibration curve was linear over the 10–50 μg/ml concentration range with a correlation coefficient of 1.0000. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.29 μg/ml and 0.89 μg/ml, respectively. The accuracy and the precision of the developed method were significantly good (RSD &lt; 2%). The validity of the proposed method was further confirmed through the statistical comparison of the obtained data with those of the official method.

DETERMINATION OF AFLATOXIN M1 IN RAW MILK PRODUCE AT YOBE STATE UNIVERSITY FARM DAMATURU, NIGERIA

The study was carried out to determine the contamination of aflatoxin M1 (AFM1) in samples (n=66) of raw milk, from three distinctive animal species (cow, n = 30; goat, n = 20; sheep, n = 16) at Yobe State University farm Damaturu in 2018. The analytical strategy utilized was high-performance liquid chromatography (HPLC). Immunoaffinity columns were used to achieve clean–up step during HPLC and fluorometric determination. The outcomes demonstrated that 36 (54.54%) samples out of the 66 samples are debased with AFM1. The sullying rates of AFM1 in dairy animals, goat milk and sheep milk were 80.0%, 25.0% and 46.75% respectively. The mean concentration for the cow, goat and sheep milk was 0.1333µg/l, 0.0462µg/l and 0.0519µg/l respectively. The general mean convergence of AFM1 levels for positive samples from the three distinctive species was 0.0727 µg/l and there was no huge contrast (p = 0.3624) in fixation levels between the three species. The estimated intake (EDI) of AFM1 from consumption of cow milk products by teachers and the students was 0.00158g/kg b.w/day based on one-day recall methods, while hazard index was recorded to be 1.58 x10-4. The high levels of AFM1 concentration recorded in this study is an indication of contamination by the fungus during storage of feeds, this may have negative effects on the human and animal’s health since it’s proven to be carcinogenic, causes growth impairment and immune suppression. Measures should be enforced on the storage of feeds which will consequently decrease the odds of aflatoxin in milk of

HPLC determination of massoia lactone in fermented Cordyceps sinensis mycelium Cs-4 and its anticancer activity in vitro.

The fermentation product of Cordyceps sinensis mycelium Cs-4 was commonly used as alternative substitutes of natural C. sinensis. Massoia lactone is the dominant component in the volatile oil of Cs-4 mycelium. In this research, we present a high performance liquid chromatography (HPLC) method for the quantitation of massoia lactone in Cs-4 mycelium. The high and stable contents of massoia lactone with values of 2.98-3.77mg/g, indicated that massoia lactone could be considered as a marker for the quality assessment of this product. The results of MTT and CCK-8 assay showed that Cs-4 mycelium volatiles exhibited cytotoxicity against eight malignant tumor cells (IC50 =6.0-49.8μg/ml) in comparison to the anticancer drug 5-fluorouracil (IC50 =17.0-425.3μg/ml), and massoia lactone might be the chemical basis for the anticancer effects of Cs-4 mycelium. Compared to the commercial drugs paclitaxel and docetaxel (IC50 =253-1973μg/ml), the Cs-4 mycelium volatiles and massoia lactone were discovered to possess inhibitory to taxol-resistant cell lines (IC50 =1.5-8.6μg/ml). PRACTICAL APPLICATIONS: Considering that there is still a lack of marker components distinctive to Cs-4 mycelium, the HPLC method represents a useful tool for the quality evaluation of Cs-4 mycelium. Moreover, the volatile oil of Cs-4 mycelium and massoia lactone have prominent anticancer property in vitro. It gives a clue that Cs-4 mycelium, the volatile oil and massoia lactone could be potentially employed in the food and medical industries for its anticancer applications.

High performance liquid chromatographic determination of cyromazine and its derivative melamine in poultry meats and eggs

A high performance liquid chromatography (HPLC) with NH2 column and 75% acetonitrile eluate to determine the insecticide cyromazine residues in poultry meats and eggs was developed. Samples were treated with NaOH, extracted with acetonitrile containing 20% concentrated NH4OH, and cleaned up through C18 Sep-Pak cartridge. The peaks of cyromazine and its metabolite melamine were successfully separated within 12 min and cleanly differentiated from interfering peaks of the samples. The detection limit of both compounds was 0.02 ppm. Recoveries of cyromazine and melamine ranged from 92.8-97.3% and 91.0-96.1% at fortified levels of 0.2-0.7 ppm, respectively. Each reaction coefficient of linear standard curve of cyromazine and melamine was better than 0.999. Using this HPLC determination, only one beef sample from 46 tested samples was detected to contain 0.04 ppm cyromazine. This was further confirmed by gas chromatography-mass spectroscopy.

High performance liquid chromatographic determination of imazalil residue in agricultural products

A simple, rapid and reproducible residue analytical method for imazalil in fruits and vegetables has been developed. The method involved the use of an ion-exchange cartridge for sample clean up followed by high performance liquid chromatography with ultraviolet detector. Acetic ether was used for extraction. Bond Elut PRS and Bond Elut C18 were used for purification. The chromatography column used Inertsil ODS-3 and the mobile phase was created by acetonitrile-10 mM KH2PO3 solution (pH 2.5), in the ratio of 35:65. The detection limit is 0.01 ppm. Rape, cabbage, honeydew melon, cumquat, mango and corn were each spiked with 0.05-3.0 ppm imazalil to be compared with the recovery study. The yield was 88.7-97.9% for rape, 72.5-87.4% for cabbage, 81.4-90.5% for honeydew melon, 81.9-87.2% for cumquat, 75.0-85.3% for mango, and 86.8-99.0% for corn. When the method was applied in analyzing commercial fruits and vegetables, no imazalil residue was detected in 20 selected samples.

Solid-phase extraction and high performance liquid chromatographic determination of steroids adulterated in traditional Chinese medicines

A systematic evaluation and comparison of the recoveries of eight steroids from spiked traditional Chinese medicine (TCM) extract using silica gel solid-phase extraction (SPE) are reported. The eight steroids, betamethasone, cortisone acetate, dexamethasone, hydrocortisone acetate, methylprednisolone, prednisolone, prednisone and triamcinolone, adulterated in TCM were assayed simultaneously by high performance liquid chromatography on a reverse phase column. This chromatographic method was carried out using isocratic elution with acetonitrile and water (3:7). Fludrocortisone acetate was used as an internal standard and detection was at 240 nm. Calibration curves of the eight adulterants were constructed in the range 6-96 μg/mL. Their correlation coefficients ranged from 0.9983 to 0.9996. The relative standard deviations of the eight steroids for intraday and interday analyses were 0.7-4.1% and 1.0-4.4%, respectively. Optimized experimental conditions including solvent composition, TCM extract concentration and different brands of SPE cartridge were also investigated.

High Performance Liquid Chromatographic Determination of Anthocyanin Pigments in Some Varieties of Black Rice

Anthocyanin pigments in ten pigmented rice cultivars were isolated with cellulose thin-layer chromatography and preparative high performance liquid chromatography (HPLC), and further identified with conventional chromatographic techniques. Individual anthocyanins were then quantified by HPLC using a reversed ODS-5 column and detected at 280 nm. Ten rice varieties contained two major anthocyanins, cyanidin 3-glucoside and peonidin 3-glucoside. Total anthocyanin contents varied greatly in the range of 0-493 mg/100 g grain among varieties. Cyanidin 3-glucoside, an agent having important oxygen radical absorbing capacity (ORAC), was most abundant in Suwon #415 variety (470 mg/100 g grain), while Jawangdo (10 mg/100 g grain) and Ilpumbyeo (not detected) had the smallest amount of anthocyanin among the samples examined. As for the distribution ratio of individual anthocyanin, Suwon #415 was characterized by a relatively high percentage of cyanidin 3-glucoside (95.3%); whereas, Suwon #425 and Heugjinmi were characterized by a relatively larger amount of peonidin 3-glucoside.

HPLC determination of 9,10-anthraquinone-2-carboxylic acid in serum and its application to pharmacokinetic study

A simple and sensitive high-performance liquid chromatographic (HPLC) method involving UV detection was developed for the determination of 9,10- anthraquinone-2-carboxylic acid (AQCA) in rabbit serum. A reverse-phase column (Lichrosher ®100RP-18) was eluted with a mobile phase of 0.4% phosphoric acid: acetonitrile (7:3) / methanol = 45 : 55 at a flow rate of 1.2 mL/min. The UV absorbance was monitored at 256 nm. After a simple clean- up procedure, the limit of quantitation achieved was 0.6 μg/mL and the standard curve was found to be linear over the serum concentration of 0.6 - 18μg/mL. The intra-assay and inter-assay coefficient of variance in serum was less than 2%, and the recovery was 96.98%. The established HPLC method was applied to the study of pharmacokinetics and bioavailability of 9,10- anthraquinone-2-carboxylic acid (AQCA). Eighteen healthy rabbits were divided into three groups and given intravenous (i.v.), oral and rectal administrations of different AQCA preparations with a single dose of 10 mg/kg. Blood samples were collected and AQCA concentrations in serum were analyzed. The results were performed with Winnolin programs and the ANOVA test (α = 0.05) was used to compare the pharmacokinetic parameters of AQCA in the three regimens. For the i.v. route, the t( 1/2 ) and AUC were 4.43 ± 0.13 hrs and 141.79 ± 7.84 μg*hr/mL, respectively. Parameters for the oral route were: T(max) = 7.17 ± 0.41 hrs; t( 1/2 ) = 15.32 ± 1.86 hrs; MRT = 20.02 ± 2.07 hrs and AUC = 107.53 ± 4.50 μg*hr/mL. The oral route indicated a slower absorption rate, a longer residence time and a lower extent of the AQCA absorption. For the rectal route, T(max) occurred at 1.58 ± 0.20 hrs, the t( 1/2 ) was 5.67 ± 0.82 hrs and the AUC was 121.18 ± 6.19 μg*hr/mL. The results indicated that the rate and extent of AQCA rectal absorption were better compared to those of the oral route. At this dose, the absolute bioavailabilities of AQCA were 0.876 and 0.872 for oral and rectal administrations, respectively. The differences of these pharmacokinetic parameters might be due to the physicochemical properties of AQCA in different conditions.

Hydrophobic deep eutectic solvent as a green extractant for high-performance liquid chromatographic determination of tetracyclines in water samples.

A green extractant, hydrophobic deep eutectic solvent was first introduced for extraction of tetracycline, oxytetracycline, and chlortetracycline from environmental water samples prior to high-performance liquid chromatography determination. Deep eutectic solvents consist of methyltrioctylammonium chloride and various medium-chain alcohols/acids, and are easy in preparation, low cost and toxicity, desirably biodegradable, and biocompatible. Theoveralltimerequired for sample preparation was 6min and the volume of organic solvent used for extraction was only 400µL. Under the optimized extraction condition, the present method yielded low limit of detection (0.5-2.0ng/mL), acceptable precision (relative standard deviations < 9.7%), good linearity from 2.0 to 500ng/mL (r2 ≥0.9991). This optimized procedure was applied for determination of tetracyclines in different water samples with desirable spiked recovery ranged from 77.5 to 87.6%. There is, therefore, a great potential to further expand application of the method for investigation of other ultra-trace analyte(s) in environmental matrixes.