The high-performance liquid chromatography and detection of phospholipids and triglycerides: Part 1. Nonpolar stationary phase chromatographic behavior in ultraviolet transparent mobile phases

The liquid chromatographic retention behavior of triglyceride and phospholipid molecular species in the nonpolar stationary (reverse) phase mode is presented for mobile phases compatible with ultraviolet detection. Interpretation of the results by means of the Kovats retention index suggests that correlation of retention to structure requires information on the number of acyl double bonds contained within the lipid molecular species in question. This, in turn, indicates that detection plays a major role in elucidating lipid structure because insufficient information is contained in the chromatographic retention data.     

Determination of glycerol, 1,2-propanediol and triglycerides by high-performance liquid chromatography and a post-column reactor containing immobilized glycerol dehydrogenase

A fluorimetri method is described for the determination of glycerol, 1,2-propanediol and triglycerides in serum by high-performance liquid chromatography with an on-line post-column reactor containing immobilized glycerol dehydrogenase. Before separation, triglycerides are cleaved with lipase and esterase. The polyhydric alcohols are separated from each other on a Finepak SIL C₁₈ (10 μm) column with water as eluent. The NADHI produced from the enzymatic reaction is monitored by fluorimetry. Calibration curves are linear between 0.01 mM and 1.0 mM for glycerol or 2.0 mM for 1,2-propanediol. The method gave satisfactory results for control sera.     

Development of a selective post-column detector for phenols separated by high-performance liquid chromatography

An on-line post-column reactor based on air-segmented continuous flow is described for the determination of phenols. The reaction used is the coupling of diazotized sulfanilic acid with phenols to form highly colored azo dyes. The effect of experimental parameters on the detector response was inveestigated by both univariate and simplex approaches in order to establish optimum reaction conditions. The aqueous reaction system is compatible with common reverse-phase solvents. The detection limit for phneol with the derivatization detector (71 μg 1^?1 shows a 16-fold improvement over u.v. detection of the underivatized phenol. Imprecision, based on multiple injections of sample into the HPLC system and measurement of the peak heights, is ± 0.64% (RSD). The technique is applied to the determination of phenols added to river water and present in residual fuel oil samples.     

Analysis of alkyl organoiodide mixtures by high-performance liquid chromatography using electrochemical detection with a post-column photochemical reactor

A method for the analysis of six alkyl organoiodides (iodomethane, iodoethane, 1-iodopropane, 1-iodobutane, 1-iodopentane, 1-iodohexane) commonly found in acetic acid process was developed. In this method the target analytes were determined by high-performance liquid chromatography (HPLC) using a post-column photochemical reactor with electrochemical detection (ED) in less than 30 min. HPLC was performed in ODS C18 reversed-phase column (5 microm, 250 x 4.6 mm I.D.) under isocratic conditions with methanol-0.067 M acetate buffer (70:30, v/v), pH 6.2 as mobile phase at flow-rate 1.1 ml/min. Alkyl organoiodides, which are electrochemically inactive, were made oxidizable at potential of 120 mV after post-column irradiation with low-pressure mercury lamp in a knitted PTFE tube. The photoreactor was placedin an aluminum housing full of nitrogen in order to prevent from the interference of oxygen. The detection limit for most analytes was of the order of 1-2 microg/l. The HPLC-ED method with a post-column photochemical reactor has good precision and linearity and can be readily applied to the routine determination of alkyl organoiodides in real acetic acid samples.     

Determination limits in high-performance liquid chromatography of plant phenolic compounds with an ultraviolet detector

The capabilities of high-performance liquid chromatography (h.p.l.c.) for the determination of phenolic compounds in 80% ethanol extracts from plant material are described. A reversed-phase column was used and elution was done with a linear gradient from 0.01 M phosphoric acid up to methanol. The efficiency of the method was studied via determination limits, defined as the minimum concentration of a compound (μg of compound per gram of extracted dry plant material) necessary to provide 90% probability that the relative error on the determination of the compound in an extract from a plant sample taken at random is < 10%. These limits take into account matrix interferences as a source of error, and were calculated with a minicomputer for the determination of 19 phenolic compounds in plant extracts. For good determinations, the concentrations of the components should be in the range 1–10 mg g^?1 of dry plant material. Separating the extracts into different chemical groups (on ion-exchange materials) prior to h.p.l.c. decreases the determination limits about five times. The dependence of determination limits on the u.v. characteristics of the compound, the sample clean-up, and the column characteristics are discussed quantitatively by means of a simple empirical equation.     

Determination of Thiamethoxam residues in honeybees by high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor

A method for the determination of Thiamethoxam in bee samples was set up by means of high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor (HPLC-h nu-ED). Analytical method was based on a rapid sample extraction procedure with acetone, followed by chromatographic separation into a C18-RP column isocratically operated by 60 mM phosphate buffer/acetonitrile (75/25) mobile phase at pH 2.7. A photochemical reactor was used as a tool to verify and eventually quantify the presence of Thiamethoxam in the samples by distinguishing it from interference contribution. Detection was performed with a potential of 880 mV after a photoactivation with a 254 nm light. The least detectable dose was 0.002 mg kg(-1). Recovery rates ranged between 59.88 and 71.62%.     

Measurement of bromate in bread by liquid chromatography with post-column flow reactor detection

This method is suitable for the determination of bromate residues in a variety of baked goods. The peer-verified method trial was performed on white bread, multigrain bread, and coffee cake spiked with known levels of potassium bromate. The analytical portion is extracted with deionized water to remove bromate from the bulk of the baked product. The aqueous extract is carried through a series of steps to remove co-extractives that would interfere with the liquid chromatography (LC) in the determinative step or hasten the deterioration of the LC column. The extract is filtered before passing it through a reversed-phase solid-phase extraction (SPE) column and a cation-exchange column in the silver form to remove lipids and chloride, respectively. Ultrafiltration is then used to remove proteins with molecular weights of >30,000 daltons. Finally, a cation-exchange column in the sodium form is used to remove silver ions from the extract. The determinative step uses LC with a reversed-phase column and an ion-pairing agent in the mobile phase. Detection is based on the post-column reaction of bromate with o-dianisidine to form an oxidation product that is quantitated spectrophotometrically at 450 nm. Overall agreement between the submitting and peer laboratories was quite good. For bromate levels of 10-52 ppb, overall mean recoveries were 76.9 and 78.8% for the submitting and peer laboratories, respectively. The standard deviations were higher for the results of the peer laboratory, probably because of the generally higher level of baseline noise present in the chromatograms. The results demonstrate that the method provides adequate accuracy with low-fat as well as high-fat foods. Bromate at levels as low as 5 ppb (ng/g) can be detected with the method.     

Metallic copper-containing post-column reactor for the detection of thiram and disulfiram in liquid chromatography

A reaction detector has been developed for the selective detection of thiram and disulfiram. The detection is based on the post-column complexation of these analytes on a solid-state reactor packed with finely divided metallic copper to form a coloured copper complex, copper(II) N,N-dimethyldithiocarbamate, with an absorption maximum at 435 nm. The method is combined with a pre-concentration and clean-up step on a pre-column to permit the sub-ppb determination of, e.g., thiram in surface water samples or disulfiram in urine. Separation is achieved by reversed-phase liquid chromatography.     

A post-column photochemical detector for use in the determination of trace metals with n-butyl-2-naphthylmethyldithiocarbamate by high-performance liquid chromatography

The determination of traces of metals by high-performance liquid chromatography of their n-butyl-2-naphthylmethyldithiocarbamate (BNMDTC) complexes on a nonpolar stationary phase, with a post-column photochemical detector is described. Metal complexes of this ligand are thermodynamically stable and kinetically inert towards dissociation. To provide low detection limits, a photochemical reactor was developed to generate a fluorescent product. A major product of the photolysis of BNMDTC complexes is identified as the highly fluorescent n-butyl-2-naphthyl-methylamine. Detection of metal ions as BNMDTC complexes at the 10^?8 M level is feasible. Iron, nickel, mercury and cobalt complexes are readily determined.     

Simultaneous multichannel mass-specific detection for high-performance liquid chromatography using an array detector sector-field mass spectrometer

The use of a separation step, such as liquid chromatography, prior to inductively coupled plasma mass spectrometry (ICP–MS) has become a common tool for highly selective and sensitive analyses. This type of coupling has several benefits including the ability to perform speciation analysis or to remove isobaric interferences. Several limitations of conventional instruments result from the necessity to scan or pulse the mass spectrometer to obtain a complete mass spectrum. When the instrument is operated in such a non-continuous manner, duty cycle is reduced, resulting in poorer absolute limits of detection. Additionally, with scanning instruments, spectral skew can be introduced into the measurement, limiting quantitation accuracy. To address these shortcomings, a high-performance liquid chromatograph has been coupled to an ICP–MS capable of continuous sample introduction and simultaneous multimass detection. These features have been realized with a novel detector array, the focal plane camera. Instrument performance has been tested for both speciation analysis and for the elimination of isobaric interferences. Absolute limits of detection in the sub picogram to tens of picograms regime are obtainable, while the added mass dimension introduced by simultaneous detection dramatically increases chromatographic peak capacity.     

Determination of organic peroxides by liquid chromatography with on-line post-column ultraviolet irradiation and peroxyoxalate chemiluminescence detection

A combination of the electrophoretically mediated microanalysis methodology with a partial filling technique was applied for the inhibition study of bovine liver rhodanese by 2-oxoglutarate. In this set-up, part of the capillary is filled with the best buffer for the enzymatic reaction, while the rest of the capillary is filled with the optimal background electrolyte for separation of substrates and products. The estimated value of K_I for 2-oxoglutarate was 3.62·10⁻⁴±1.43·10⁻⁴ M with respect to cyanide and 1.40·10⁻³±1.60·10⁻⁴ M with respect to thiosulfate. In addition, the type of inhibition was also evaluated. The findings of 2-oxoglutarate as the competitive inhibitor with respect to cyanide and as the uncompetitive inhibitor with respect to thiosulfate are in accordance with previous literature data.     

Determination of organic mercury species in soils by high-performance liquid chromatography with ultraviolet detection.

Reversed-phase high-performance liquid chromatography with ultraviolet detection was optimized for the simultaneous separation and quantification of nine organic mercury compounds: methyl-, ethyl-, phenyl-, methoxyethyl-, ethoxyethyl-, benzoic and tolylmercury, mersalylic acid and nitromersol. The nine compounds were successfully separated on octadecylsilane columns (200 x 3 mm i.d.) by gradient elution with a methanol-water mixture ranging from 30 to 50% v/v. The detection limits for the various compounds are in the range 7.0-95.1 micrograms dm-3. For the extraction of five organomercurials from spiked soils, eight different extraction solutions were tested to differentiate between the total content and the available/soluble fraction of the analytes. Ammonium acetate solutions (1 mol dm-3) and water proved to be suitable agents for the estimation of the available and soluble fractions of methyl-, ethyl-, benzoic, methoxyethyl- and ethoxyethylmercury. For the determination of the total content of methyl- and benzoic mercury in soils, solutions of potassium iodide (1 mol dm-3)-ascorbic acid (0.1 mol dm-3) and oxalic acid (1 mol dm-3) provided recoveries in the ranges 53-81%. None of the solutions tested is suitable for the extraction of ethyl-, methoxyethyl- and ethoxyethylmercury.     

Determination of cetirizine in serum using reversed-phase high-performance liquid chromatography with ultraviolet spectrophotometric detection.

A method using reversed-phase high-performance liquid chromatography with ultraviolet detection for the determination of ceterizine in serum is described. The method is sensitive down to 50 ng/ml (250-microliter loop). Sample preparation involves only serum deproteination with perchloric acid and injection of the centrifuged supernatant. Elution is at pH 2.5 with acetonitrile-methanol-0.05 M phosphate buffer (33:9:58, v/v) on a 25 cm x 4.6 mm I.D. Spherisorb S5 ODS2 column. Detection is at 211 nm, its lambda max. For levels above 300 ng/ml the serum sample size is 100 microliter and a 200-microliter sample is necessary for concentrations less than 300 ng/ml. At the 2 micrograms/ml concentration the intra-assay relative standard deviation is better than 2.2%, whilst the inter-assay deviation is 2.6% over eight samples. At 200 ng/ml the intra-assay relative standard deviation is 6% over seven samples. Detector response is linear from 100 ng/ml to 10 micrograms/ml (100-microliter loop).     

Determination of H2O2 and organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection

A method for the determination of hydrogen peroxide and several organic peroxides by high-performance liquid chromatography with post-column UV irradiation, derivatization and fluorescence detection is described. By means of post-column UV irradiation in the presence of water organic peroxides are converted into hydrogen peroxide and organic hydroperoxides, which react rapidly with the post-column derivatization agent p-hydroxyphenylacetic acid (PHPAA) under catalysis of horseradish peroxidase to yield the fluorescent PHPAA dimer that is detected at excitation and emission wavelengths of 285 and 400 nm, respectively. The detection limit for hydrogen peroxide is 14 ng/mL, for organic peroxides between 34 ng/mL and 5 μg/mL. No interference by other compounds was observed when their concentrations were below 10 mg/mL except ethers and phenols. Received: 6 August 1997 / Revised: 11 December 1997 / Accepted: 15 December 1997