Development of a capillary zone electrophoresis method for caseinoglycomacropeptide determination

Caseinoglycomacropeptide (CGMP) is a polypeptide of 64 amino acid residues, derived from the C-terminal part of bovine κ-casein. A sensitive and selective capillary zone electrophoresis method has been developed and validated for the analysis and quantitation of CGMP. Separation is carried out at 30 kV, using an uncoated fused-silica capillary and 20 mM sodium citrate buffer at acidic pH 3.5. The described method allows the separation of various CGMP subcomponents. The validation data proves that the method has the requisite selectivity, sensitivity, reproducibility and linearity for CGMP assay and for quality control during CGMP manufacturing (batch-to-batch reproducibility).     

Development of a method for the determination of cefovecin in plasma by HPLC

A simple high‐performance liquid chromatography method for the determination of cefovecin in small volume plasma has been developed. Following solid‐phase extraction using Oasis HLB cartridges, samples were separated by reverse‐phase high‐performance liquid chromatography on an XBridge C₈ (3.5 µm) 4.6 × 250 mm column and quantified using ultraviolet detection at 280 nm. The mobile phase was a mixture of 10 mm ammonium acetate (pH 3.5) and acetonitrile (89:11), with a flow rate of 0.85 mL/min. The standard curve ranged from 0.1 to 200 µg/mL. Intra‐ and Inter‐assay variability for cefovecin was <10%, and the average recovery was >90%. The lower limit of quantitation was 0.1 µg/mL. This method was successfully applied to the analysis of cefovecin samples at our institution. This is also the first fully validated method with an internal standard that does not use mass spectrometry. Copyright © 2014 John Wiley & Sons, Ltd.     

Development of a new high-performance liquid chromatographic method for the determination of ceftazidime

A rapid, accurate, and sensitive high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of ceftazidime in pharmaceuticals. The method validation parameters yielded good results and included range, linearity, precision, accuracy, specificity, and recovery. The excipients in the commercial powder for injection did not interfere with the assay. Reversed-phase chromatography was used for the HPLC separation on a Waters C18 (WAT 054275; Milford, MA) column with methanol-water (70 + 30, v/v) as the mobile phase pumped isocratically at a flow rate of 1.0 mL/min. The effluent was monitored at 245 nm. The calibration graph for ceftazidime was linear from 50.0 to 300.0 microg/mL. The values for interday and intraday precision (relative standard deviation) were <1%. The results obtained by the HPLC method were calculated statistically by analysis of variance. We concluded that the HPLC method is satisfactory for the determination of ceftazidime in the raw material and pharmaceuticals.     

Development of a luminol-based chemiluminescence flow-injection method for the determination of dichlorvos pesticide

A simple, fast chemiluminescence (CL) flow-injection method based on the reaction of luminol with H₂O₂ in the presence of a cationic surfactant (cyltrimethylammonium bromide, CTMAB) has been described for the direct determination of dichlorvos pesticide (DDVP). Under the optimal conditions, the CL intensity was linear to the DDVP concentration in the range of 0.02–3.1 μg ml⁻¹ (r=0.9998, n=10). The relative standard deviation was 3.4% at 0.35 μg ml⁻¹ (n=10), with a detection limit (3σ) of 0.008 μg ml⁻¹ DDVP. The possible reaction mechanism was also discussed. This method has been successfully applied to the determination of trace DDVP residue in vegetable sample and results have been compared with that of the UV method.     

Development of a rapid method for the quantitative determination of deoxynivalenol using Quenchbody

Quenchbody (Q-body) is a novel fluorescent biosensor based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. In order to develop a method using Q-body for the quantitative determination of deoxynivalenol (DON), a trichothecene mycotoxin produced by some Fusarium species, anti-DON Q-body was synthesized from the sequence information of a monoclonal antibody specific to DON. When the purified anti-DON Q-body was mixed with DON, a dose-dependent increase in the fluorescence intensity was observed and the detection range was between 0.0003 and 3 mg L⁻¹. The coefficients of variation were 7.9% at 0.003 mg L⁻¹, 5.0% at 0.03 mg L⁻¹ and 13.7% at 0.3 mg L⁻¹, respectively. The limit of detection was 0.006 mg L⁻¹ for DON in wheat. The Q-body showed an antigen-dependent fluorescence enhancement even in the presence of wheat extracts. To validate the analytical method using Q-body, a spike-and-recovery experiment was performed using four spiked wheat samples. The recoveries were in the range of 94.9–100.2%. The concentrations of DON in twenty-one naturally contaminated wheat samples were quantitated by the Q-body method, LC-MS/MS and an immunochromatographic assay kit. The LC-MS/MS analysis showed that the levels of DON contamination in the samples were between 0.001 and 2.68 mg kg⁻¹. The concentrations of DON quantitated by LC-MS/MS were more strongly correlated with those using the Q-body method (R² = 0.9760) than the immunochromatographic assay kit (R² = 0.8824). These data indicate that the Q-body system for the determination of DON in wheat samples was successfully developed and Q-body is expected to have a range of applications in the field of food safety.     

Graphical method for the determination of the complex NMR shift and equilibrium constant for a hetero-association accompanying self-associations

We describe a novel graphical method which, in conjunction with the previously proposed graphical determination of monomer shift, dimer shift, and dimerization constant for self-association, allows us to determine the complex shift and equilibrium constant for a hetero-association, A + B ⇌ AB, accompanying self-associations, A + A ⇌ A₂, and B + B ⇌ B₂. The merit of the new method includes the removal of the restrictions imposed on the conventional Benesi-Hildebrand (B-H) plot: (1) that the concentration of one component must be much less than that of the other; (2) that there be no accompanying self-association. The simultaneous equilibrium of the self-association of 2-pyrrolidone (A) and that of 4-methyl-α-pyrrolidone (B) and the hetero-association between A and B in acetonitrile-d₃ at 25‡C is studied. The inappropriateness of the B-H plot in dealing with this case is also pointed out.     

氨基酸铜螯合率测定光度新方法的研究

研究了新试剂对羧基偶氮安替比林的合成及其与铜的显色反应,建立测定氨基酸铜螯合率的光度方法。在乙酸介质中,Cu2 与对羧基偶氮安替比林反应形成组成比为2∶1的蓝色配合物,配合物的最大吸收峰位于622nm,表观摩尔吸光系数达3.03×104L·mol-1·cm-1,Cu2 的质量浓度在0～0.80μg/mL符合比耳定律。此外,显色体系有很高的选择性,大多数金属离子允许量在mg级以上。氨基酸铜螯合率的测定包括结合态和游离态铜的分析,由于在氨基酸铜的水溶液中加入对羧基偶氮安替比林试剂时,氨基酸铜被试剂立即分解并转化为稳定的对羧基偶氮安替比林铜配合物,因此,总铜量的测定不需消化样品而直接进行。游离态的铜经葡聚糖凝胶(G-15)吸附后,用稀HCl洗脱后按上述方法测定。     

Development of a method for the determination of 226Ra by liquid scintillation counting

Liquid scintillation counting has not been widely applied to a-particle detection because of its poor energy resolution and variable background. In the present work, a time saving and reasonably accurate method for determination of ²²⁶Ra in water has been developed, using liquid scintillation spectrometry and pulse-shape analysis. The effect of three levels of chemical quench on the spillover of alpha interactions into the beta window and vice versa was assessed. The advantages of liquid scintillation in comparison with other methods (radon emanation) for determination of ²²⁶Ra are the high counting efficiency (~100%) and the easier sample preparation, with no need for sample preconcentration.     

Development of a capillary electrophoresis method for the separation of flavonolignans in silymarin complex

CE method for the baseline separation of structurally similar flavonolignans silybin A, silybin B, isosilybin A, isosilybin B, silychristin, silydianin, and their precursor taxifolin in silymarin complex has been developed and validated. The optimized background electrolyte was 100 mmol/L boric acid (pH 9.0) containing 5 mmol/L heptakis(2,3,6-tri-O-methyl)-β-CD and 10% (v/v) of methanol. The separation was carried out in an 80.5/72 cm (50 μm id) fused silica capillary at +25 kV with UV detection at 200 nm. Genistein (10 μg/mL) was used as internal standard. The resolution between the diastereomers of silybin and isosilybin was 1.73 and 2.59, respectively. The method was validated for each analyte in a concentration range of 2.5–50 μg/mL. The calibration curves were rectilinear with correlation coefficients ≥0.9972. The method was applied to determine flavonolignans in two dietary supplements containing Silybum marianum extract. The accuracy was evaluated by comparing the results of the CE analyses of the dietary supplements with those of the reference United States Pharmacopeial HPLC method. The unpaired t-test did not show a statistically significant difference between the results of both the proposed CE and the reference method (p > 0.05, n = 3).     

Development of a solid-phase extraction method for the determination of polychlorinated biphenyls in water

Polychlorinated biphenyls (PCBs) in water were extracted with a rebuilt extraction unit using 47 mm C18 solid-phase extraction (SPE) disks. Three types of disks (SPEC, ENVI and Empore) were investigated for the extraction of seven PCBs from 11 reagent water spiked at two concentration levels (20 and 1000 ng/l). The Empore disks produced the best analyte recoveries (91-107% with R.S.D. of 1-8%) at the low concentration level and displayed no leaking tendency. Empore disks were therefore considered superior to ENVI and SPEC disks for the conditions outlined in this work. The obtained extracts were dried and purified in an additional clean-up step using custom-made columns containing Florisil and Na2SO4. For water containing large amounts of organic matter, a pre-filtration was included. Final analysis was carried out on a dual-column GC-electron-capture detection system with on-column injection. The optimised extraction method, including clean-up, was less time-consuming and used less hazardous organic solvents than conventional liquid-liquid extraction (LLE) methods. Recoveries were 92-102% with R.S.D. of 3-8%.     

Development of a novel potentiometric titration method for determination of polypropylene degradation

In this study, a novel potentiometric titration of hydroperoxide in degraded polypropylene (PP) is proposed. This titration is quite sensitive compared with the conventional ones such as UV and manual titrations, and its detection limit was about 2 meq/kg. The sensitivity was equal to that of molecular weight measurement by GPC for the degraded PP and, in addition, the volatilization behavior of the hydroperoxide could be detected. This titration was found to be very effective for the determination of PP degradation.     

Development and prevalidation of a method for phenol determination by solid-phase spectrophotometry

A simple, sensitive and rapid solid-phase spectrophotometric procedure was developed for the determination of traces of phenol with 4-aminoantipyrine as a reagent (AAP-SPS), and the optimal experimental conditions were established. This method was performed by sorption and direct absorbance measurements of the product phenol-AAP sorbed on the anion-exchanger Dowex 1-X4 (0.2 g) at 495 nm (absorption maximum) and 700 nm (non-absorption wavelength). The sensitivity offered by the AAP-SPS procedure was higher by a factor of 40 compared with the respective conventional spectrophotometric method. Metrological characteristics were established using a prevalidation strategy. The AAP-SPS procedure is characterized by a linear calibration function in the working range of 0.05–0.50 μmol, low standard deviation of procedure (±0.012), low limit of determination (0.021 μmol), and favorable random (±0.85 to ±11.27%) and systematic deviations (−4.55 to +11.50%). Moreover, the accuracy of the system investigated by the recovery test is acceptable (99–102%). Favorable working and performance characteristics make the new SPS method ideal for phenol monitoring in pharmaceutical preparations as well as other matrices.     

Chemometrics-assisted spectrophotometric method for simultaneous determination of vitamins in complex mixtures

An advanced independent component analysis algorithm (MILCA) is applied for simultaneous chemometric determination of fat- and water-soluble vitamins in complex mixtures. The analysis is based on the decomposition of spectra of multicomponent mixtures in the UV region. The key features of the proposed method are simplicity, accuracy, and reliability. Comparisons between the new algorithm and other established methods (MCR-ALS, SIMPLISMA, other ICA techniques) were made. Our results indicate that in most cases, MILCA is comparable or even outperforms other chemometrics methods taken for comparisons. The influence of different factors (abundance of components, noise, step of spectral scan, and scan speed) on decomposition performance has been investigated. The optimal conditions for spectroscopic registration have been identified. The proposed method was used for analysis of model mixtures and real objects (multivitamin drugs, food additives, and energy drinks). The resolved concentrations match well with the declared amounts and the results of reference methods.     

Orthophosphate determination: A simple spectrophotometric method for the measurement of the orthophosphate-triethylamine-molybdate complex

Summary Triethylamine is a selective precipitant for phosphomolybdic acid which allows separation of orthophosphate from phosphoric acid esters and anhydrides. In this paper a simple method for the estimation of the orthophosphate-triethylamine-rnolybdate complex, based on its ultraviolet absorption spectrum, is described. The precipitate, corresponding to a range of 5–250 nmole of orthophosphate, is dissolved in 3 ml of methanol and the absorbance of the solution measured at 320 nm against water. The molar absorptivity of the complex at this wavelength is 2.09 × 10⁷1·mole^–1·cm^–1.

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Orthophosphat-Bestimmung: Eine einfache spektrophotometrische Methode zur Bestimmung des Orthophosphat-Triäthylamin-Molybdat-Komplexes

Zusammenfassung Triäthylamin ist ein selektives Fällungsreagens für Phosphormolybdänsäure, das die Trennung von Orthophosphat aus Mischungen von Phosphorsäureestern und Anhydriden erlaubt. Eine einfache Methode zur Bestimmung von Orthophosphat-Triäthylamin-Molybdat wurde beschrieben. Sie beruht auf der Messung der Ultraviolett-Absorption des Komplexes. Fünf bis 250 nMol gefälltes Orthophosphat werden in 3 ml Methanol gelöst und die Absorption dieser Lösung gegen Wasser bei 320 nm gemessen. Der molare Extinktionskoeffizient des Komplexes unter diesen Bedingungen ist 2,09·10⁷ 1·Mol^–1·cm^–1.

     

Development and validation of a SPE-GC-MS method for the determination of pesticides in surface water

A method for analysis of 20 commonly used pesticides in surface water based on solid-phase extraction and gas chromatography-mass spectrometry was proposed. During method development the key parameters that can affect SPE extraction and determination such as selection of efficient SPE sorbent, pH of water sample, type and volume of elution solvent, breakthrough volume and matrix effects were investigated. The method was validated using spring water spiked with appropriate concentration of pesticides. The obtained correlation coefficients were in range 0.9972–1.000, limits of detection (LOD) were 0.001–0.5?µg?L^?1 and the limits of quantification (LOQ) were 0.005–1?µg?L^?1 depending on a pesticide. Much higher LOD (20?µg?L^?1) and LOQ (50?µg?L^?1) values were obtained for bentazone. The influence of matrix was assessed using real water samples spiked with appropriate concentration of pesticide standards solution. Both signal enhancement and suppression were observed, depending on a pesticide, therefore standard addition method was used for pesticides determination. The developed method was applied on real water samples taken in close vicinity of agricultural fields. Many of the targeted pesticides were found in the samples and the results are presented in this article.     

Development and validation of a capillary electrophoretic method for the determination of amiodarone and desethylamiodarone

Summary A simple, sensitive and rapid capillary electrophoretic method has been developed for the separation and quantification of amiodarone and its metabolite, desethylamiodarone. The compounds were separated in a capillary of 45 cm effective length and 75 μm i.d., by use of an applied voltage of 25 kV and an electrolyte containing 15mm ADA buffer (pH 7.5), 10mm SDS, and 70% (v/v) acetonitrile. The selectivity, precision, linearity, range, sensitivity, and robustness of the method were good. The applicability of the assay was demonstrated by analyzing these drugs in serum. Electrokinetic injection with field-amplified sample-stacking was used to increase sensitivity. The limit of detection of the serum assay was 6.46 ng mL⁻¹ and the precision 3.7%.     

Development of a method for the complex isolation of physiologically active components from bee venom

A method is proposed for isolating the main components (melittin, apamin, and phospholipase A₂ from the venom of the bee Apis mellifera using HPLC, and the synthesis of two sorbents for fractionating these peptides is described.A. S. Sadykov Institute of Biooganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 62 70 71. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 862–866, November-December, 1995. Original article submitted March 13, 1995.     

Development and validation of a UHPLC method for the determination of flavonoids in red wine

A simple and fast ultra-high-performance liquid chromatography (UHPLC) method was developed for the identification and quantification of the following flavonoids in red wine: (+/-)-catechin, (-)-epicatechin, rutin, quercitrin, hesperidin, neohesperidin, (+/-)-naringenin, hesperetin, and chrysin. Chromatographic separation of the flavonoids was performed on a Chromolith Fast Gradient C18e column. A gradient elution was used with mobile phases consisting of 0.1% formic acid in water and acetonitrile. UV detection was performed at 280 nm. A complete separation of flavonoids was possible within 6 min. The calibration curves showed good linearity (R2 > or = 0.9990) in the selected range of each analyte; the LOD ranged between 0.06 and 0.19 microg/mL. An optimized sample preparation method utilized SPE. The Oasis HLB column with the highest recoveries was selected for the preconcentration step. This method was successfully applied to the determination of these flavonoids in the red wine samples with excellent results.