Liquid Chromatogaphy Research Articles

Stabilitas Vitamin D3 pada Dadih (Susu Kerbau Fermentasi)

Vitamin D deficiency and inadequacy is a worldwide problem that can cause serious health problems. Vitamin D reduce the risk of osteoporosis, cancer, diabetes mellitus, and cardiovascular disease. Vitamin D is obtained from four sources such as sun exposure, food, supplements and fortification food. The research was conducted in October 2019. The purpose of this study was to see the stability of vitamin D3 in dadih during storage. The High Performance Liquid Chromatogaphy (HPLC) method was used to calculate vitamin D3 levels in fortified dadih. Quantification of vitamin D3 was done on day 0, day 3, day 9, day 16, day 23 and day 30. Statistical analysis showed that there was no significant difference in the level of vitamin D3 in dadih for 30 days of storage although there was a decrease in the level of vitamin D3 in dadih on the fourth week but it was not significantly (p> 0.05). In conclusion, Vitamin D3 in the dadih was stable for 30 days of storage although there was a decrease in vitamin D3 levels on the 16th and 30th days of storage due to the undetectable vitamin D3 in some samples. The addition of vitamin D3 to dadih is suggested using encapsulation techniques to increase solubility, longer shelf life, prevention of chemical degradation, better dispersibility, higher bioavailability and effectiveness.

Supercritical fluid chromatography using methacrylate-based monolithic column for the separation of polar analytes.

A poly(N,N-dimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl) ammonium betaine-co-ethylene dimethacrylate) monolith was prepared in a 100× 2.0mm id stainless-steel column and was investigated for supercritical fluid chromatography. Optimization of its porosity was performed by changing the conditions of polymerization. Then, the chemical group of this column was confirmed by Fourier transform infrared spectroscopy and elemental analysis. The morphology was characterized by scanning electron microscopy. The prepared column showed good repeatability based on the retention factor of adenine, uracil and cytosine to calculate their run-to-run, day-to-day, column-to-column, and batch-to-batch relative standard deviations. Those values were less than 1.9% (n=10), 6.5% (n=3), 5.6% (n=3), and 1.7% (n=3), respectively. In addition, the column was found to be stable within 3 and 10 days with relative standard deviations less than 6.5 and 8.5%, respectively. These results indicated that the columns showed good reproducibility and stability. Compared with liquid chromatogaphy, supercritical fluid chromatography provided better kinetic performance and selectivity. Finally, several neutral, acid, and basic polar analytes were utilized to test its application. The results demonstrated that the prepared column exhibited a good separation performance and showed great potential in supercritical fluid chromatography.

In vitro Dissolution Method for Alendronate Sodium and Vitamin D3 Tablets Using HPLC with Combination of Refractive Index and Ultraviolet Detectors

Two separate HPLC methods were developed for the In-vitro dissolution of Alendronate sodium and Vitamin D3 tablets using Ultra-violet and Refractive index detection. Dissolution method for the estimation of Alendronate sodium (Method-I) in the fixed dose combination uses High Performance Liquid Chromatogaphy (HPLC) with Refractive Index detector (RID) whereas dissolution method for Vitamin D3 (Method-II) uses HPLC with Ultraviolet detector. Method-I uses Hamilton PRP-X500 (150 x 4.6) mm, 7 µ column with 0.01 % Trifluoro acetic acid in water, pH adjusted to 2.5 as mobile phase at a flow rate of 1.5 mL min−1 with Refractive detector sensitivity of 256. Method-II uses Zorbax SB CN (150 x 4.6) mm, 5 µ column with a mixture of water, methanol and acetonitrile in the ratio of 20:72:8 v/v/v as mobile phase at a flow rate of 1.2 mL min−1 with Ultraviolet (UV) detector wavelength of 266 nm. The method was validated for specificity, linearity, precision, accuracy and robustness as per International Conference on Harmonization (ICH) guidelines. This method is useful for the In-vitro dissolution of Alendronate and Vitamin D3 (Cholecalciferol) tablets during routine analysis in Quality control laboratory.

Quantification of individual polysulfides in lab-scale and full-scale desulfurisation bioreactors

Environmental context Emission into the atmosphere of gaseous streams containing sulfur compounds, such as H2S and SOx, will lead to the unwanted formation of acid rain. In order to prevent this, biological processes can be employed to treat sulfur-containing gas streams. In this study, we describe a way to investigate the speciation of polysulfide anions in biodesulfurisation systems, which might enable further understanding and development of these processes. Abstract Environmental pollution caused by the combustion of fuel sources containing inorganic and organic sulfur compounds such as hydrogen sulfide (H2S) and thiols, is a global issue as it leads to SO2 emissions. To remove H2S from gas streams such as liquefied petroleum gas (LPG), biological processes can be applied. In these processes, polysulfide anions (Sx2–) play a significant role as they enhance the dissolution of H2S and act as intermediates in the biological oxidation of hydrogen sulfide ions to elemental sulfur. Despite their important role, the distribution of the various polysulfide species in full-scale biodesulfurisation systems has not yet been reported. With conventionally applied spectrophotometric analysis it is only possible to determine the total concentration of Sx2–. Moreover, this method is very sensitive to matrix effects. In this paper, we apply a method that relies on the derivatisation of Sx2– to dimethyl polysulfanes. Owing to the instability of higher dimethyl polysulfanes (Me2S4 to Me2S8), standards are not commercially available and had to be prepared by us. We present a simplified quantification method for higher dimethyl polysulfanes by calculating high performance liquid chromatogaphy (HPLC) UV response factors based on the addition of internal standards. The method was subsequently used to assess the distribution of polysulfide anions in both a laboratory-scale and a full-scale biodesulfurisation unit. We found that the average chain length of polysulfides strongly depends on the process conditions and a maximum of 5.33 sulfur atoms per polysulfide molecule was measured. Results of this study are required by mechanistic and kinetic models that attempt to describe product selectivity of sulfide oxidising bioreactors.

Development of a novel switchable CE-MS interface with predictive trajectories for high-throughput proteomics studies

Abstract The capillary electrophoresis (CE)-mass spectrometry (MS) interface is anticipated to have pivotal roles to play in biomarker discovery, elucidation and validation, and in diagnostics, as well as in drug discovery. We are developing a CE-MS system as the natural extension of our Label-Free Intrinsic Imaging (LFiI™) platform, allowing a large increase in analytical power. One of the most powerful analytical tools in bio-analytical science applications is the mass spectrometer, allowing identification and extraordinary resolving power. However, this instrument too has shortcomings – it really needs a separation system ahead of it. Traditionally liquid chromatogaphy (LC) or 2D-gel separation have been coupled to MS with relative robustness. However LC techniques are not ideally suited to the analysis of biomolecules, with the use of denaturing solvents, expensive columns etc. while flat gel systems have serious shortcomings in sensitivity, reproducibility, dynamic range, quantification and throughput. These are largely overcome with capillary approaches, such as the Label Free Intrinsic Imaging system, the Peregrine, developed by deltaDOT Ltd of London. The CE-MS interface being developed by deltaDOT QSTP (Qatar Science and Technology Park) will be based on our real-time pattern recognition known as ‘predictive trajectories’ (‘PT’) and biomolecular switching on the LFiI platform, to allow specific protein bands, selected ‘on the fly’, to be separated and quantified, before they undergo trypsin digestion, injection and full MS analysis in a Waters high-definition mass spectrometer (‘Synapt’). Such an interface considerably reduces the system noise by giving a switch opportunity to analyse selected bands of interest from the bulk of background buffer and highly concentrated proteins of low interest. Another significant advantage of such an approach would arise from a more coherent signal where peptide fragments of the same protein would elute together, as opposed to traditional liquid chromatography where any peptide fragments could come from any proteins from the sample, requiring heavy and slow analyses processing. Based on work in our QSTP labs, we present preliminary data of analyses undertaken with partners in Qatar University on a range of proteomic targets and other analytes. We show how the unique combination of LFiI + PT + HDMS Q-Tof can provide a significant enhancement in analytical power.

Relationship between urinary phytoestrogen levels and Z score of lumbar vertebral bone mineral density in Japanese postmenopausal women

Phytoestrogens are attracting much attention for their anticancer effects, their reduction of menopausal symptoms, and their preventive effects against osteoporosis in the human body, resulting from the consumption of foods that contain phytoestrogens at high concentrations. The effects of 2 phytoestrogens, genistein and daidzein, have been reported, but little data have been published on the effects of equol, coumesterol, and other phytoestrogens. In the present study, using high-performance liquid chromatogaphy, we measured the urinary phytoestrogen levels of 80 postmenopausal Japanese women and simultaneously determined the bone mineral density (BMD) in lumbar vertebrae L2 through L4 (L2-L4BMD), measured the levels of the bone biochemical markers and calciotropic hormones, and also studied the relationships of these with urinary phytoestrogens. Our results showed the following values for phytoestrogens in grams per milligram of creatinine: daidzein, 3.11 ± 3.09; genistein, 2.74 ± 5.02; equol, 6.29 ± 11.44; coumesterol, 0.51 ± 0.77; O-desmethylangolensin (DMA), 1.59 ± 5.19; formononetin, 0.11 ± 0.19; and biochanin A, 0.08 ± 0.19. When these were converted to logarithms, log (genistein) was the only one to show a statistically significant positive correlation ( r = 0.29, P < .05) with the Z score of L2-4BMD. Log (DMA) showed a negative correlation ( r = −0.36, P < .013) with the urinary deoxypyridinoline level. The levels of osteocalcin and the calciotropic hormones showed no correlation with urinary phytoestrogen levels. The fact that there were correlations between the genistein level and Z score of bone density, and between DMA and biochemical markers of bone metabolism, suggested that phytoestrogens may have an effect on bone metabolism in Japanese women. Genistein and DMA might be involved in the inhibition of bone resorption in terms of the correlation between phytoestrogens levels and BMD.

Detailed characterisation of sulphur and nitrogen components in a multifunctional, ashless lubricant additive by spectroscopic and chromatographic techniques

AbstractThe work reported here used infrared (IR), multinuclear nuclear magnetic resonance (NMR), and chromatographic techniques to identify and characterise a commercial sulphur‐ and nitrogen‐containing ashless multifunctional additive. The proper assignment of signals in the 1H/13C NMR and IR spectra of the sample has facilitated the identification of different types of sulphur‐ and nitrogen‐containing components. The methodology involves investigation by NMR and IR spectroscopies, thin‐layer chromatography (TLC) and high‐performance liquid chromatogaphy (HPLC) techniques to elucidate the types of components present. This requires the generation of NMR and IR spectral data for standard compounds of alkyl sulphides with different sulphur content and alkyl chain, and nitrogen and sulphur components such as thiadiazole, imidazoline, triazole, etc., and spectral comparison with the spectra of the unknown sample. Further, these components have been separated by silica/alumina column chromatographic and preparative TLC techniques and subsequently analysed for their exact chemical structure by spectroscopic techniques. The combined spectroscopic and chromatographic analyses have yielded the presence of four types of components: di‐t‐octyl polysulphide, di‐t‐octyl thiadiazole, a component containing amine ether functionality, and alkyl amine salts of mono/dialkyl phosphoric acid.

2,3-Naphthalimidoacetyl Chloride as a Labeling Reagent for Aliphatic Amines and Alcohols in Liquid Chromatography

A new, highly reactive and sensitive labeling reagent, 2,3-naphthalimidoacetyl chloride, has been synthesized and used to determine aliphatic amines and alcohols using high-performance liquid chromatogaphy. Primary and secondary amines can be completely labeled with the reagent within 1 min in acetonitrile at room temperature in the absence of and in the presence of triethylamine, respectively. Reactions of the reagent with aliphatic primary and secondary alcohols proceed to completion within 10 min in benzene at room temperature in the presence of 4-dimethylaminopyridine. The derivatives could be readily eluted with acetonitrile:water (90:10). The detection limits (signal-to-noise ratio = 3) with fluorescence detection were 4 fmol (λ ex = 258 nm, λ em = 391 nm).

Investigation of the metabolism of felodipine using on‐line liquid chromatography/UV diode array detection/atmospheric pressure chemical ionization mass spectrometry as a diagnostic tool

AbstractThe utility of on‐line liquid chromatogaphy coupled to UV diode array detection and atmospheric pressure chemical ionization mass spectrometry for the analysis of microsomal metabolites of felodipine has been investigated. A previously unreported metabolite of felodipine, extracted from male rat microsomes, has been detected using this technique. UV diode array detection has been used to characterize this metabolite as a dihydropyridine metabolite and tandem mass spectrometry data used to assign a structure.

Direct coupling of micro high-performance liquid chromatogaphy with fast atom bombardment mass spectrometry : III. Application to oligosaccharides

Description du systeme d'ionisation, d'analyse et de l'interface en ligne. Mise en application pour le glucose, le lactose, le raffinose, le maltotriose et quelques pentoses et hexoses. Interpretation des spectres de masse

Chromatographic analysis of fatty acid dimers : Comparison of gas—liquid chromatography, high-performance liquid chromatography and thin-layer chromatography with flamce ionization detection

For monitoring fatty acid polymerization and isolatioin processes, a sufficiently rapid and reproducible analytical procedure is still lacking. This paper compares the results of the analysis of mixtures of monomeric and oligomeric fatty acids by means of gas—liquid chromatography, high-performance liquid chromatogaphy, and thin- layer chromatography with flame ionization detection. For serial analyses, the last mentioned method appeared to be the most suitable.

Determination of free and total catecholamines and salsolinol in urine by ion-pair reversed-phase liquid chromatography with electrochemical detection after a one-step sample clean-up

A simple method is presented for the routine determination of free and total catecholmines and salsolinol in urine. For the analysis of total catecholamines and salsolinol, the urine samples are first hydrolysed. The compounds are isolated by a one-step sample clean-up by cation-exchange chromatography and separated by ion-pair reversed-phase liquid chromatogaphy and detected electrochemically. The method is suited for routine analysis. Pretreatment of ten samples for the high-performance liquid chromatographic analysis of free and total catecholamines and salsolinol is carried out in 4 h. A single analysis is completed in 30 min. The absolute losses of the compounds were less than 10%, except for epinephrine (20%). The detection limits were 0.9, 2.4, 4.8 and 6 nmol/1 for norepinephrine, epinephrine, dopamine and salsolinol, respectively, in native urine. The values were twice as high for hydrolysed urine. The sample clean-up appears to be rather specific: apart from catecholamines and salsolinol, only those structurally related mines that contain a vicinal hydroxyl group are isolated.

Determination of serum Δ 5-3β-hydroxysteroid sulphates by combined high-performance liquid chromatography and immobilized 3β,17β-hydroxysteroid dehydrogenase in column form

We studied the use of an immobilized enzyme, covalently bound to aminopropyl-CPG, in the analysis of individual Δ 5-3β-hydroxysteroid sulphates. A microcolumn with immobilized 3β,17β-hydroxysteroid dehydrogenase was prepared and used together with high-performance liquid chromatogaphy (HPLC). The reduced nicotinamide-adenine dinucleotide produced from Δ 5-3β-hydroxysteroids by this enzyme was fluorimetrically determined. The immobilized enzyme was sufficiently stable for at least one month or for 180 tests when used repeatedly. A clinical trial demonstrated that this HPLC—immobilized enzyme method is superior to the soluble enzyme method, giving reliable and reproducible results at a low cost.

Determination of penicillin G and cloxacillin residues in beef and pork tissue by high-performance liquid chromatography

Tissues were homogenized and then deproteinized with acetonitrile. The acetonitrile extract was partitioned between dichloromethane and pH 2.2 buffer and then extracted wih pH 7 buffer. After addition of ammonium sulfate to the aqueous solution, it was mixed with acetonitrile. The acetonitrile extract was separated and evaporated, and the residue was taken up in water. The aqueous solution was analyzed by high-performance liquid chromatogaphy (HPLC) on a C 18, 10-μm particle size, reversed-phase column using gradient elution with 0.01 M orthophosphoric acid—acetonitrile (from 80:20 to 40:60 in 20 min) at a flow-rate of 1 ml/min and UV absorbance detection at 220 nm. Recoveries were generally greater than 90% with all tissues. Data on incurred residues from a treated cow showed recoveries of penicillin which were frequently several times higher by HPLC than by bioassay. Sensitivity limits in muscle were about 0.05 ppm for both penicillin G and cloxacillin, but higher in liver and kidney because of interferences. The method is suitable for other monobasic penicillins but not for dibasic or amphoteric penicillins.

Decomposition of the herbicide EPTC in soils with a history of previous EPTC applications

Abstract The degradation of the herbicide EPTC (S-ethyl N, N-dipropylthiocarbamate) in 3 soils, with and without, previous field exposure to this herbicide was investigated. The influence of storage of soil (12 months) on the subsequent breakdown of EPTC was investigated. Soils with added EPTC were incubated (22°C), and the fate of EPTC was followed using gas liquid chromatogaphy. The herbicide degraded more rapidly in soils with 3 and 4 years of previous' exposure to annual applications of EPTC (6kg/ha) than in those soils without any previous exposure. This more rapid decomposition was evident after storage of soils for I year at ambient temperatures. Microorganisms were responsible for EPTC degradation in these soils. Some of the implications of this phenomenon on soil ecology are discussed.

Chemical characterization of cyanogen bromide fragments from the β-chain of human complement factor C3

The isolated β-chain of human complement factor C3 (C3β) was fragmented by cyanogen bromide. Nine fragments were defined by gel filtration and high-pressure liquid chromatogaphy, and characterized with respect to their Mr, amino acid composition and N-terminal amino acid sequence. Approx. 30% of the primary structure of C3β was determined. Alignment of the 3 N-terminal fragments allowed determination of 61 of the amino terminal residues of C3β. This region demonstrated 40% homology with the sequence in the N-terminal segment of the α-chain of the cobra venom factor.

A new excitation method in laser fluorometry and Raman spectroscopy using an optical fiber - An application to high performance liquid chromatography.

The use of an optical fiber as a wave guide for a laser beam reduces the undesired scattered light in trace fluorometry and in Raman spectroscopy of microliter-size samples. It also permits effective sample excitation and easy optical alignment. In conjunction with high performance liquid chromatogaphy (HPLC), this excitation method has improved the detection limit by an order of magnitude without any other change in the conventional HPLC fluorometric detection system.

Simultaneous determination of hippuric acid and o-, m- and p-methylhippuric acids in urine by high-performance liquid chromatogaphy

Simultaneous determination of hippuric acid and o-, m- and p-methylhippuric acids in urine by high-performance liquid chromatogaphy

Measurement of progabide and its deaminated metabolite in plasma by high-performance liquid chromatogaphy and electrochemical detection

Progabide (4-{[4-chlorophenyl-5-fluoro-2-hydroxyphenol)-methylene]amino}butanamide) and its deaminated metabolite were measured simultaneuosly in plasma by high-performance liquid chromatography. Both compounds were extracted from plasma and the molecules were stabilized at the methylene—amino double bond with sodium borohydride reduction. Oxidative electrochemical detection was used for final quantitation. The method was used to measure progabide and progabide acid in plasma from a healthy volunteer who received a single 1200-mg dose of progabide orally. Lower limits of detection for progabide and progabide acid were 30 and 15 ng/ml, respectively. Coefficient of variation was less than 5% for both compounds.

Cellular uptake and metabolism of daunorubicin as determined by high-performance liquid chromatogaphy : Application to L1210 cells

The metabolism of daunorubicin (DNR) to daunorubicinol and daunomycinone in murine leukaemia cells has been examined by means of high performance liquid chromatography. A rapid and efficient extraction method has been developed that permits the recovery of the drug and its metabolites from cell homogenates. By means of high-performance liqud chromatography daunorubicinol and daunomycinone have been separated from DNR and the intracellular concentration of the compounds determined. The method developed is very rapid and sensitive, and amounts a small as 30 pg of DNR can be detected. The results indicate that the aldo-keto reductase is not very active in L1210 cells in culture, the main intracellular product found being DNR.