Standard Chromatographic Method Research Articles

Determination of Underivatized Long Chain Fatty Acids Using HPLC with an Evaporative Light‐Scattering Detector

AbstractA high‐performance liquid chromatographic method with an evaporative light‐scattering detector has been developed for the separation and quantitative analysis of four underivatized long chain fatty acids in four different oil matrices. An isocratic elution mode using methanol/water/acetic acid and an Agilent Eclipse XDB‐C18 analytical column was used. Calibration curves of the four fatty acids (FA) were well correlated (r2 > 0.999) within the range of 1–10 mg mL−1 for linoleic acid, 0.8–10 mg mL−1 for stearic acid and 0.5–10 mg mL−1 for the other FA. Four oil samples were examined; camellia oil, olive oil, Brucea javanica oil and sesame oil. Good agreement was found with the standard gas chromatographic (GC) method. The proposed method offers distinct advantages over the official GC method; better separation and precision, and the sample components do not need to be derivatized.

An Ultraviolet-Spectrophotometric Method for the Determination of Glimepiride in Solid Dosage Forms

Considering the cost of acquiring a liquid chromatographic instrument in underdeveloped economies, the rising incidence of diabetes mellitus, the need to evaluate the quality performance of glimepiride generics, and the need for less toxic processes, this research is an imperative. The method was validated for linearity, recovery accuracy, intra- and inter-day precision, specificity in the presence of excipients, and inter-day stability under laboratory conditions. Student's t test at the 95% confidence limit was used for statistics. Using 96% ethanol as solvent, a less toxic and cost-effective spectrophotometric method for the determination of glimepiride in solid dosage forms was developed and validated. The results of the validated parameters showed a λ(max) of 231 nm, linearity range of 0.5-22 μg/mL, precision with relative SD of <1.0%, recovery accuracy of 100.8%, regression equation of y = 45.741x + 0.0202, R(2) = 0.999, limit of detection of 0.35 μg/mL, and negligible interference from common excipients and colorants. The method was found to be accurate at the 95% confidence limit compared with the standard liquid chromatographic method with comparable reproducibility when used to assay the formulated products Amaryl(®) (sanofi-aventis, Paris, France) and Mepyril(®) (May & Baker Nigeria PLC, Ikeja, Nigeria). The results obtained for the validated parameters were within allowable limits. This method is recommended for routine quality control analysis.

A high performance liquid chromatographic method for determination of the radiochemical purity of iodohippuric acid [123I] injection.

A method for the separation of the radiopharmaceutical iodohippuric acid ([123I]IHA) from the two impurities produced in the labelling reaction, iodide (123I-) and iodobenzoic acid ([123I]IBA) has been developed using reversed-phase high performance liquid chromatography (h.p.l.c.). The effect of eluent pH on the separation of the three compounds was studied. An eluent consisting of methanol: 0.1 M acetic acid (30:70) adjusted to pH 4.0 with NaOH gave optimum phase capacity ratios of -0.21 for I-, 0.93 for IHA and 1.54 for IBA. On the chromatograph used, this corresponded to an analysis time of 4 min per sample. To recover 123I- from the column with a good peak shape, it was found necessary to include sodium iodide (1 mg/100 ml) in the eluent. The recoveries of 123I-123IHA and 123IBA from the column were found to be 100.6% (s.d. 1.3), 100.2% (s.d. 2.1) and 98.1% (s.d. 1.2) respectively (n = 10 for each compound). H.p.l.c. was compared with the standard thin layer chromatographic (t.l.c.) method of analysis on 10 preparations of [123I]IHA which gave a mean radiochemical purity of 99.1% (+/- 0.2 s.e.) by h.p.l.c. and 98.3% (+/- 0.2 s.e.) by t.l.c. This difference was found to be significant (P less than 0.05) and an explanation for this difference is proposed. A coefficient of variation of 0.1% on the results from the repeated analysis (n = 20) of one preparation of [123I]IHA, demonstrated that the h.p.l.c. method was capable of very high precision.

Alkylation or Silylation for Analysis of Amino and Non-Amino Organic Acids by GC-MS?

Gas chromatography–mass spectrometry (GC-MS) is a widely used analytical technique in metabolomics. GC provides the highest resolution of any standard chromatographic separation method, and with modern instrumentation, retention times are very consistent between analyses. Electron impact ionization and fragmentation is generally reproducible between instruments and extensive libraries of spectra are available that enhance the identification of analytes. The major limitation is the restriction to volatile analytes, and hence the requirement to convert many metabolites to volatile derivatives through chemical derivatization. Here we compared the analytical performance of two derivatization techniques, silylation (TMS) and alkylation (MCF), used for the analysis of amino and non-amino organic acids as well as nucleotides in microbial-derived samples. The widely used TMS derivatization method showed poorer reproducibility and instability during chromatographic runs while the MCF derivatives presented better analytical performance. Therefore, alkylation (MCF) derivatization seems to be preferable for the analysis of polyfunctional amines, nucleotides and organic acids in microbial metabolomics studies.

Alkylation or Silylation for Analysis of Amino and Non-Amino Organic Acids by GC-MS?

Gas chromatography–mass spectrometry (GC-MS) is a widely used analytical technique in metabolomics. GC provides the highest resolution of any standard chromatographic separation method, and with modern instrumentation, retention times are very consistent between analyses. Electron impact ionization and fragmentation is generally reproducible between instruments and extensive libraries of spectra are available that enhance the identification of analytes. The major limitation is the restriction to volatile analytes, and hence the requirement to convert many metabolites to volatile derivatives through chemical derivatization. Here we compared the analytical performance of two derivatization techniques, silylation (TMS) and alkylation (MCF), used for the analysis of amino and non-amino organic acids as well as nucleotides in microbial-derived samples. The widely used TMS derivatization method showed poorer reproducibility and instability during chromatographic runs while the MCF derivatives presented better analytical performance. Therefore, alkylation (MCF) derivatization seems to be preferable for the analysis of polyfunctional amines, nucleotides and organic acids in microbial metabolomics studies.

Determination of underivatized long chain fatty acids using RP-HPLC with capacitively coupled contactless conductivity detection

A reversed-phase high-performance liquid chromatographic method with capacitively coupled contactless conductivity detector (C(4)D) has been developed for the separation and the simultaneous determination of five underivatized long chain fatty acids (FAs), namely myristic, palmitic, stearic, oleic, and linoleic acids. An isocratic elution mode using methanol/1mM sodium acetate (78:22, v/v) as mobile phase with a flow rate of 0.6 mL min(-1) was used. The separation was effected by using a Hypersil ODS C(18) analytical column (250 mm x 4.6 mm x 5 microm) and was operated at 45 degrees C. Calibration curves of the five FAs were well correlated (r(2)>0.999) within the range of 5- 200 microg mL(-1) for stearic acid, and 2-200 microg mL(-1) for the other FAs. The proposed method was tested on four vegetable oils, i.e., pumpkin, soybean, rice bran and palm olein oils; good agreement was found with the standard gas chromatographic (GC) method. The proposed method offers distinct advantages over the official GC method, especially in terms of simplicity, faster separation times and sensitivity.

Determination of average carbon number of petroleum waxes by X-ray diffraction

Eight petroleum waxes, both paraffin as well as microcrystalline, have been analysed by X-ray diffraction. The average carbon number has been estimated by the long-range ordering observed in the diffractograms of these waxes. The average carbon number has also been determined following the standard gas chromatographic (GC) method. The results obtained by X-ray diffractometry compare well with those obtained by the GC method. The former method also permits determination of the average carbon number of high melting point waxes, which is otherwise difficult using GC.

Evolving Window Zone Selection method followed by Independent Component Analysis as useful chemometric tools to discriminate between grapefruit juice, orange juice and blends

This study investigates the use of high resolution 1H NMR as a suitable alternative to the standard chromatographic method for the determination of adulteration of orange juice ( Citrus sinensis) with grapefruit juice ( Citrus paradisi) based on flavonoid glycoside content. Fifty-nine orange juices (OJ), 23 grapefruit juices (GJ) and 10 blends (OG), obtained from local retail outlets were used to assess the performance of the 1H NMR method. The work presented here introduces the Evolving Window Zone Selection (EWZS) function that holds promise for the automatic detection of spectral regions tailored to discriminate predefined groups. This technique was applied on the pre-processed 1H NMR spectra of the 92 juices. Independent Component Analysis (ICA) is a good alternative to Principal Component Analysis (PCA) for recovering linearly-mixed unobserved multidimensional independent signals and has been used in this study to build supervised models that classify the samples into three categories, OJ, GJ, OG. The regions containing the known flavonoid glycoside markers were selected as well as another zone containing the signals of sucrose, α-glucose and other components that were tentatively attributed. ICA was applied on three different groups of selected variables and showed good results for both discrimination and interpretation of the signals. Up to 97.8% of the juices were correctly attributed. This method gave better results than the commonly used PCA method. In addition, the time required to carry out the 1H NMR analysis was less than half the time of the standard chromatographic method.

PARAFAC2 and MCR-ALS quantification of Diltiazem antihypertensor based on a kinetic spectrophotometric methodology

A Diltiazem kinetic spectrophotometric UV–Vis method, based on a reaction of the Diltiazem with hidroxylamine and a ferric salt, was used for the quantification of Diltiazem in different pharmaceutical formulations. This method is based on the acquisition of three-way data structures [wavelength (nm) × time (s) × concentration (mg/L)] followed by chemometric analysis by an appropriate PARAFAC2 or MCR-ALS second-order calibration model. The results obtained are compared with those obtained by direct determination, at maximum wavelength, and by the United States Pharmacopeia (USP) standard chromatographic method. For all the pharmaceutical formulations analysed good quantification results were found with PARAFAC2 and MCR-ALS second-order calibration models. For bulk drug analysis, detection limits of 6 and 2 mg/L, and for pharmaceutical formulations analysis, an average detection limit of 41 and 39 mg/L were found, respectively with PARAFAC2 and MCR-ALS.

Henry's Law Coefficients for Monomers and Selected Solvents in Amorphous Tetrafluoroethylene−Hexafluoropropylene Copolymers

This paper presents Henry's law coefficients for tetrafluoroethylene (TFE) and hexafluoropropylene (HFP) monomers, their dimers, and a number of potential process solvents in amorphous TFE−HFP copolymers. A standard inverse gas chromatographic method was used for the measurements. Results are presented to validate the experimental techniques and data reduction methods by comparison with published literature values for polystyrene with selected solvents. The specific amorphous TFE−HFP copolymer used for these measurements was characterized by fluorine NMR as consisting of 53 mol % HFP and 47 mol % TFE with 15% of the HFP as diads. A number-average molecular weight of 104 000 with a polydispersity of 2.81 was determined by gel permeation chromatography analysis. A total of 19 binary systems were measured for this polymer at 4−6 temperatures over the range of (321 to 511) K. The experimental data are correlated by fitting with an Arrhenius temperature dependency.

Quadruple-pulsed amperometric detection for simultaneous flow injection determination of glucose and fructose

A quadruple-potential waveform, at E det1=−0.5 V ( t det1=240 ms), E det2=+0.2 V ( t det2=180 ms), E oxd=+1.0 V ( t oxd=180 ms), and E red=−0.8 V ( t red=300 ms) versus SCE, was employed at a Nafion-coated Au electrode for the simultaneous determination of glucose and fructose by flow injection analysis. E det1 and E det2 caused the oxidation of glucose and glucose and fructose, respectively. Hence, concentration subtraction could be used to determine both species. Using the FI-PAD, a linear response to glucose at E det1 was obtained across the concentration range 5–60 mM, with a sensitivity of 2.34×10 −7 A mM −1, and the limit of detection of 1.2 mM (S/N=3). Whereas at E det2 scaled linearly with either glucose or fructose concentration in the range up to 60 mM, with a sensitivity of 7.50×10 −6 A mM −1, and the limit of detection of 0.13 mM (S/N=3). Interference from other sugars, organic acids, and amino acids were studied. The method developed was used to analyze glucose and fructose contents in five different fruit samples; the method showed good agreement with the standard liquid chromatographic method.

Detection of verapamil drug by fluorescence and trilinear decomposition techniques

A three-way analytical methodology experimentally based on fluorescence excitation emission matrix (EEM) and in PARAFAC and TLD chemometric analysis was assessed for the quantification of verapamil drug in a tablet formulation. A standard addition procedure generates experimental information compatible with the chemometric data analysis model allowing the estimation of verapamil with a detection limit of about 0.04mg/l using methanol as solvent. The structure of the verapamil EEM follows a trilinear model, but background signals (first- and second-order scatter bands) did not—a trilinear three-factor model is necessary to describe experimental datasets. The comparison of a three-factor PARAFAC model with a United States Pharmacopoeia (USP) standard chromatographic method showed similar results.

Pulsed amperometric detection of furan compounds in transformer oil

The failure of high voltage transformers can result in significant cost and supply implications to both power supplier and consumer alike and in extreme cases may result in explosion, serious injury or death. Transformer failure can be predicted by measuring furanics present in the oil, produced by the thermolytic breakdown of cellulosidic insulators. Failing units can have furanic levels of up to 10 μg ml −1. The use of pulsed amperometric detection (PAD) to measure furanics in transformer oils in real time is reported here. Oils were examined by pre-extraction or direct suspension in aqueous measurement solution or by solubilisation and direct PAD measurement in organic solvents. Linear relationships between PAD response and furanic concentration was found for 2-furaldehyde and furfuryl alcohol (F-OH) across the range of 0–10 μg ml −1, with PAD proving most sensitive to the latter compound. PAD was performed directly in the organic phase in t-butanol with 0.1 M tetramethyl ammonium hydroxide, with aged oils containing >2 μg ml −1 of 2-furaldehyde yielding data within close agreement (<9%) of a standard chromatographic method. The simplicity and rapidity of this method offers the power transmission industry a means of monitoring furanic levels in transformers in real time, thereby reducing the risk of uncontrolled transformer failure.

Determination of morphine in water immiscible process streams using sequential injection analysis coupled with acidic permanganate chemiluminescence detection†

A procedure for the determination of morphine in non-aqueous, water immiscible, process streams by sequential injection analysis is presented. Detection was based upon the chemiluminescence reaction of morphine with acidic potassium permanganate in the presence of sodium polyphosphate, which was carried out in a heterogeneous reaction environment. The calibration graph (range 0.005–0.12% m/v) was non-linear, with a polynomial equation of best fit of y = –384x2 + 139x – 4.84 (R2 = 0.9990), where y represents the singal (mV) and x represents the morphine concentration (% m/v), and was analytically useful over the range 0.01–0.1% m/v. Precision (as measured by relative standard deviation) was 6.0% for 10 replicate analyses of a non-aqueous morphine standard (0.075% m/v).Methyltriphenylphosphonium permanganate was investigated as an alternative chemiluminescent reagent and was found to deliver a similar analytical performance to potassium permanganate. Sequential injection analysis coupled with chemiluminescence detection afforded results for the determination of morphine in non-aqueous process samples which were in good agreement with those obtained using a standard liquid chromatographic method, with an analytical throughput in excess of 120 h–1.

Gas Chromatographic Determination of Polychlorinated Biphenyls in Mussels from Galicia, Spain

Abstract We have modified a standard capillary column gas chromatographic method (U.S. Environmental Protection Agency No. 505) for the determination of polychlorinated biphenyl (PCB) residues and applied the technique to mussels (Mytilus galloprovincialis) from Galicia, Spain. During the purification step, SepPak Florisil minicolumns are used instead of conventional columns. Method performance is good (limit of detection, 0.032 μg/kg; recovery, 99%; method precision as coefficient of variation among 10 samples, 2.5%), and compared with the standard method, less eluant is required and analysis is faster. The mean PCB content of 107 mussels collected from various points on the coast of Galicia was 113 μg/kg.

Determination of the anticonvulsant felbamate and its three metabolites in brain and heart tissue of rats

An automated, internal standard high-performance liquid chromatographic method for the simultaneous quantitation of felbamate and its three metabolites in adult and neonatal rat brain and heart tissue homogenates was developed and validated. The homogenates prepared from one part of the tissue and four parts of water were extracted with ethyl acetate, and the extract was evaporated to dryness and redissolved in mobile phase. Separation was accomplished on a Waters Resolve C 18, 5 μm, 300 mm × 3.9 mm I.D. column with a mobile phase consisting of 0.01 M phosphate buffer, pH 6.8—acetonitrile—methanol (800:150:50, v/v/v). Eluting peaks were monitored with an ultraviolet detector at 210 nm. The linear range of the assay for felbamate and the metabolites was 0.20–50.00 μg/ml of homogenate or 1–250 μg/g of brain or heart tissue. The lower limit of quantitation for all four analytes was 0.20 μg/ml of homogenate or 1.00 μg/g of tissue.

Organic compounds in the Forest Vale, H4 ordinary chondrite

We have analyzed the H4 ordinary chondrite Forest Vale for polycyclic aromatic hydrocarbons (PAHs) using two-step laser mass spectrometry (L 2MS) and for amino acids using a standard Chromatographic method. Indigenous PAHs were identified in the matrices of freshly cleaved interior faces but could not be detected in pulverized silicates and chondrules. No depth dependence of the PAHs was found in a chipped interior piece. Amino acids, taken from the entire sample, consisted of protein amino acids that were nonracemic, indicating that they are terrestrial contaminants. The presence of indigenous PAHs and absence of indigenous amino acids provides support for the contention that different processes and environments contributed to the synthesis of the organic matter in the solar system.

Determination of Carbon Monoxide in Blood by Gas Chromatography Using a Thermal Conductivity Detector

A method is described for the gas chromatographic quantitation of carbon monoxide by means of thermal conductivity detection. Carbon monoxide is released from blood samples as small as 0.02 mL using a unique extraction chamber. The method was compared to a standard gas chromatographic and spectrophotometric method of carbon monoxide quantitation. It was comparable to the former with all samples evaluated and apparently more reliable than the latter with decomposed samples.