Gradient Elution High-performance Liquid Chromatography Research Articles

Validation of HPLC Method for Determination of Histamine in Human Immunoglobulin Formulations.

Histamine fixed-immunoglobulin formulations, which consisted of 0.15 µg of histamine dihydrochloride and 12 mg of human immunoglobulin in a vial, are used for anti-allergic treatments, and controlling the amounts of histamine in the formulations is essential to avoid histamine intoxication. A high-performance liquid chromatography (HPLC) method for determination of histamine contents of the formulations was established and validated. Histamine extracted from the formulation was labeled with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and was analyzed by gradient elution HPLC with UV detection at 260 nm. The method showed linearity in the range 0.8-2.4 µM (R > 0.999), accuracy (100.1-105.8% recovery), and precision (relative standard deviation ≤ 1.93%). The validated method was applied for five lots of the pharmaceutical, and their histamine contents were determined to be 0.149-0.155 µg/vial. These results indicated that the validated method is useful to control amounts of histamine in biopharmaceutical products. The HPLC method was developed for quantitative determination of histamine content of the histamine fixed-immunoglobulin formulations.

Central obesity transition increased urinary levels of 8-hydroxydeoxyguanosine in male adults: A 3-year follow up study

ObjectiveAssociation of oxidative DNA damage with gain in anthropometric indices has not been fully elucidated. MethodsIn this study, participants (n = 1151) were derived from the baseline visit of Wuhan residents in the Wuhan-Zhuhai Cohort Study. The participants finished the physical examinations at both baseline and 3-year follow up. Urinary levels of 8-hydroxydeoxyguanosine (8-OHdG) were measured by gradient-elution high performance liquid chromatography method and then calibrated by urinary creatinine (Cr) values. ResultsGeneralized linear models showed that after adjusted for confounding factors, baseline central obesity individuals with a ≥2.5% hip circumference (HC) loss or >5% HC gain had a 0.290 μmol/mol Cr (95% confidence interval (CI): 0.108, 0.472) or 0.553 μmol/mol Cr (95% CI: 0.273, 0.833) increase in urinary 8-OHdG levels compared with those with a −2.5%–2.5% HC gain (both P < 0.05). Moreover, compared with non-central obesity at both baseline and 3-year follow-up, we observed that central obese men at both baseline and 3-year follow-up had a 0.46 μmol/mol Cr (95% CI: 0.16, 0.75) increased in urinary 8-OHdG levels. ConclusionsHC gain showed dose-dependent associations with urinary 8-OHdG levels. Moreover, male central obesity at both baseline and 3-year follow-up had an increased risk for urinary 8-OHdG levels.

Metabolism of Gamma-tocopherol in the Liver of Male Sprague-Dawley Rat

Vitamin E (VE) plays important roles in the physiological and biochemical processes of animals and humans. However, VE is an unique vitamin that has not been understood thoroughly because the metabolites of VE are very complicated and hard to be analysed. This experiment was conducted to study the metabolism of γ-tocopherol, one of 8 VE isomers, in the liver post-mitochondrial supernatant (S9) of male Sprague-dawley rat (Rattus norvegiens, SD-rat) using a novel approach. The results showed that an optimal reactive system of γ-tocopherol metabolism in vitro was composed of 5 mg/ml of S9, 8 μg/ml of γ-tocopherol, 1 mmol/ml of nicotine amide adenine dinucleotide phosphate (NADPH) and 45 min of reactive time at 35°C. The metabolites of γ-tocopherol could be successfully analyzed using gradient elution and reverse-phase high-performance liquid chromatography (RP-HPLC). A main metabolite was identified as ω-hydroxylate, a side chain of γ-tocopherol, when the structure of γ-tocopherol metabolites was determined using gas chromatography-mass spectrometry (GC-MS). A conclusion was drawn that ω-hydroxylate, a main metabolite of γ-tocopherol, could be most utilized by SD-rat and other mammals.

Determination of Degradation Products of Doxercalciferol by Solid-Phase Extraction and Reversed-Phase HPLC

In the current study, injectable formulations containing Doxercalciferol as the active pharmaceutical ingredient are analyzed by using gradient-elution high-performance liquid chromatography with ultraviolet detection. Various related impurities and degradants are quantified by using solid-phase extraction (SPE) for enhanced sensitivity. The assay of possible related impurities and Doxercalciferol analogues present at trace quantities is performed by using Trans-1-α-hydroxy vitamin D2 (Doxercalciferol related degradation product/Impurity B) as standard and 1-β-hydroxy vitamin D2 (Doxercalciferol related degradation product/Impurity C) as internal standards for the SPE study. The current method is shown to be stability-indicating and free from interferences from any of the formulation excipients and potential degradation products and impurities. The validated method is shown to be reproducible, accurate, sensitive and selective.

Simultaneous determination of some ultraviolet-absorbing chemicals in sunscreen cosmetics using a high-performance liquid chromatography method

A method of gradient elution high-performance liquid chromatography (HPLC) for simultaneous determination of 11 different ultraviolet-absorbing chemicals of phenylbenzlmldazole sulphonic acid, 4-aminobenzoic acid, benzophenone-4, benzophenone-3, isoamyl p-methoxycinnamate, 4-methylbenzylidene camphor, octocrylene, ethylhexyl methoxycinnamate, homosalate, ethylhexyl salicylate, methylene bis-benzotriazolyl tetramethylbutyl phenol was developed for the application to sunscreen cosmetic products. In this study, an Agilent SB-C18 analytical column (250 × 4.6 mm, 5 μm) was utilized and methanol, tetrahydrofuran and perchloric acid aqueous solution (0.2 mL HClO(4) + 300 mL H(2)O) were used for gradient elution at a total flow rate of 1.0 mL min(-1). The optimum conditions for 11 different ultraviolet-absorbing chemicals analyses were investigated. All calibration curves showed good linear regression with UV detection (311 nm) within test ranges. The correlation coefficients were better than 0.999 in all cases. The assay was simple, selective, convenient and reproducible and is suitable for the determination of ultraviolet-absorbing chemicals in commercial sunscreen cosmetic products. The use frequency of 11 different ultraviolet absorbents in 100 sunscreen cosmetics was investigated and statistically analysed. The ultraviolet absorbent of maximum use frequency was ethylhexyl methoxycinnamate.

Residue screening in apple, grape and wine food samples for seven new pesticides using HPLC with UV detection. An application to trifloxystrobin dissipation in grape and wine

A new analytical procedure was developed and validated, using liquid chromatography, for simultaneous determination of seven new pesticides belonging to different chemical classes (azoles, strobilurines, benzoylureas, and a new oxazolidinedione) in apple, grape and wine food samples. Solvent extraction of pesticides was performed using a cyclohexane-dichloromethane (9 + 1 v/v) mixture and the extract was cleaned-up by solid-phase extraction (SPE) using silica cartridges and elution with tetrahydrofurane. Separation of pesticides flusilazole, fenbuconazole, diniconazole, tebufenozide, famoxadone, trifloxystrobin and flufenoxuron was performed by gradient elution High-Performance Liquid Chromatography (HPLC) with a microbore Hypersil HS C18 column in 35 min total time, using mixtures of acetonitrile-water as the mobile phase. Pesticides were detected and quantified at 210 nm with a variable-wavelength UV detector. Recoveries of pesticides from spiked samples, at 0.05–2.0 mg kg−1 for apple and grape and at 0.025–0.250 mg L−1 for wine, ranged from 82 to 107% for apple and grape and from 87% to 106% for wine with RSD < 12%. The limits of quantification (LOQs) of the method, as a signal to noise ratio equalled 10, ranged from 0.02 to 0.10 mg kg−1 for apple and grape and from 0.005 to 0.02 mg L−1 for wine samples. The uncertainty associated with the analytical methodology, estimated using the ‘bottom-up’ approach, was lower than 12.8% for all pesticides and matrices tested. The proposed methodology was applied for the evaluation of trifloxystrobin residue levels in grapes exposed to field treatments and in the must and wine produced from them.

Identification of the major metabolites of 5‐n‐butyl‐4‐{4‐[2‐(1H‐tetrazole‐5‐yl)‐1H‐pyrrol‐1‐yl]phenylmethyl}‐2,4‐dihydro‐2‐(2,6‐dichloridephenyl)‐3H‐1,2,4‐triazol‐3‐one, a new angiotensin type 1 receptor antagonist, in rat bile by HPLC–diode array detection–MS and HPLC‐MS/MS

5-n-Butyl-4-{4-[2-(1H-tetrazole-5-yl)-1H-pyrrol-1-yl]phenylmethyl}-2,4-dihydro-2-(2,6-dichloridephenyl)-3H-1,2,4-triazol-3-one (1b), a new non-peptide angiotensin type 1 receptor antagonist, has been observed to play a positive role in the treatment of hypertension in preclinical tests. Rats were dosed with the drug, and the major metabolites in the bile were separated by gradient elution high-performance liquid chromatography. HPLC-diode array detection-mass spectrometry, HPLC-electrospray ionization MS/MS methods in negative ion mode and collision-induced dissociation were used to elucidate the structures of the major metabolites of 1b. One dihydroxylated 1b (M1), two monohydoxylated 1b (M2, M3) and one 1b monoglucuronide (M5) were found in male rat bile; however, three monohydoxylated 1b (M2, M3, M4) and one 1b monoglucuronide (M5) were found in female rat bile. These metabolites greatly differ in amount between male and female rat bile.

Development of a gradient elution high-performance liquid chromatography assay with ultraviolet detection for the determination in plasma of the anticancer peptide [Arg 6, d-Trp 7,9, mePhe 8]-substance P (6–11) (antagonist G), its major metabolites and a C-terminal pyrene-labelled conjugate

[Arg 6, d-Trp 7,9, mePhe 8]-substance P (6–11), code-named antagonist G, is a novel peptide currently undergoing early clinical trials as an anticancer drug. A sensitive, high efficiency high-performance liquid chromatography (HPLC) method is described for the determination in human plasma of antagonist G and its three major metabolites, deamidated-G (M1), G-minus Met 11 (M2) and G[Met 11(O)] (M3). Gradient elution was employed using 40 m M ammonium acetate in 0.15% trifluoroacetic acid as buffer A and acetonitrile as solvent B, with a linear gradient increasing from 30 to 100% B over 15 min, together with a microbore analytical column (μBondapak C 18, 30 cm×2 mm I.D.). Detection was by UV at 280 nm and the column was maintained at 40°C. Retention times varied by <1% throughout the day and were as follows: G, 13.0 min; M1, 12.2 min; M2, 11.2 min; M3, 10.8 min, and 18.1 min for a pyrene conjugate of G (G–P). The limit of detection on column (LOD) was 2.5 ng for antagonist G, M1–3 and G–P and the limit of quantitation (LOQ) was 20 ng/ml for G and 100 ng/ml for M1–3. Sample clean-up by solid-phase extraction using C 2-bonded 40 μm silica particles (Bond Elut, 1 ml reservoirs) resulted in elimination of interference from plasma constituents. Within-day and between-day precision and accuracy over a broad range of concentrations (100 ng/ml–100 μg/ml) normally varied by <10%, although at the highest concentrations of M1 and M2 studied (50 μg/ml), increased variability and reduced recovery were observed. The new assay will aid in the clinical development of antagonist G.

Isolation and identification of the C 6-hydroxy and C 20-hydroxy metabolites and glucuronide conjugate of methylprednisolone by preparative high-performance liquid chromatography from urine of patients receiving high-dose pulse therapy

In the present study metabolites of methylprednisolone were detected using gradient elution high-performance liquid chromatography. Separation was performed by a Cp Spherisorb ODS 5 μm (250 mm×4.6 mm I.D.) column, connected to a guard column, packed with pellicular reversed phase. The mobile phase was a mixture of acetonitrile and 1% acetic acid in water. At t=0, this phase consisted of 2% acetonitrile and 98% acetic acid 1% in water (v/v). During the following 35 min the phase changed linearly until it attained a composition of acetonitrile–buffer (50:50, v/v). At 40 min ( t=40) the mobile phase was changed over 5 min to the initial composition, followed by equilibration during 2 min. The flow-rate was 1.5 ml/min. UV detection was achieved at 248 nm. We have isolated the respective compounds with the most abundant concentration and suggested their chemical structure based on NMR, IR, UV, MS, retention behaviour and melting points. The α/β stereochemistry could not be solved in this study. The overall picture of the metabolic pathways of methylprednisolone is apparently simple: reduction of the C20 carbonyl group and further oxidation of the C20-C21 side chain (into C21-COOH and C20-COOH), in competition with or additional to the oxidation at the C6-position.

The Lichen Genus Pertusaria in the Falkland Islands (Islas Malvinas)

Eight species of the genus Pertusaria are found in the Falkland Islands (Islas Malvinas), two of which are here described as new: P. malvinae Messuti & A. W. Archer and P. salacinifera Messuti & A. W. Archer; P. alterimosa Darb., P. cerebrinula Zahlbr., P. erubescens (Hook. & Taylor) Nyl., P. macloviana Miill. Arg., P. perrimosa Nyl., and P. spegazzinii Miill. Arg. are reported from Falkland Islands. A key to the named species of Pertusaria on the Falkland Islands is given. The Falkland Islands or Islas Malvinas* are the largest island group situated in the South Atlantic Ocean, between latitudes 40o-60oS, 500 km east of the Strait of Magellan. Two main islands (West Falklands and East Falklands)* and about a hundred islets form the Falkland Islands (Cabrera 1994; Imshaug 1969). The climate of these islands is cool and moist with snow during most of the year. According to Cabrera (1994) these islands are included in the phytogeographical area named Provincia Insular (Regi6n Antlirtica o Austral, Dominio Subantirtico). The islands have been visited by a number of scientific expeditions, but few have contributed to our knowledge of the lichen flora. During the 19th century and the first years of the 20th century, many expeditions visited the Falkland Islands and collected lichens, bryophytes, and vascular plants. In 1968, the U.S. Antarctic Research Program financed an expedition that included lichenologists (H. A. Imshaug and R. C. Harris). Over 2,000 collections were made and a total of 235 species were recorded in a preliminary study (Imshaug 1969). The lichen genus Pertusaria in the Falkland Islands (Islas Malvinas) has received little attention. Until 1950, seven taxa in the genus were reported from the islands (Grassi 1950). This account is based on specimens collected by H. A. Imshaug and R. C. Harris during the 1968 expedition and on examination of the type specimens of the relevant species. The eight Pertusaria species described are all saxicolous (with the exception of P. erubescens and P. macloviana which are rarely corticolous on the Falkland Islands) and all the species lack cortical xanthones. This is unusual as, for example, P. lophocarpa Korber, which contains 4,5-dichlorolichexanthone, is found on Maquarie Island (Australia) and Campbell Island (New Zealand), both of which are in approximately the same latitude as the Falkland Islands (Archer 1997). This study has revealed a number of undescribed or poorly known taxa which are reported here. MATERIALS AND METHODS Specimens from the herbaria AAS, B, BM, CANB, CHR, CTES, EGR, ESS, G, GZU, H, H-NYL, LE, LPS, MSC, S, TU, and w were examined. Microscopy.-The techniques used for the examination of specimens have been described previously (Archer 1991). Sections of the fruiting bodies were mounted in water for measurement of the spore size. Chemistry-The chemical constituents were identified using thin layer chromatography (TLC) (Archer 1993; Culberson 1972) and gradient-elution high performance liquid chromatography (HPLC) (Elix et al. 1997; Feige et al. 1993). RESULTS AND DISCUSSION 1. PERTUSARIA ALTERIMOSA Darb., Wiss. Erg. Schwed. Sudp. Exped. IV, Liefr. 11: 7. 1912.

Deactivation of Mustard and Nerve Agent Models via Low-Temperature Microemulsions

New low-temperature oil-in-water (O/W) type microemulsions that resist freezing and phase separation at −18 °C have been developed. These systems were shown to simultaneously destroy, via oxidative and hydrolytic mechanisms, simulants of three chemical warfare agents. Reactions, monitored at 25 °C by gradient elution high-performance liquid chromatography, took place instantly or over many minutes, depending upon the particular simulant. Neglecting reaction products, the low-temperature microemulsions contained 11 components: propylene glycol, water, base, oxidant/nucleophile, surfactant, cosurfactant, oil, stabilizer, two nerve agent simulants, and a mustard simulant. Only by virtue of self-aggregation does this extraordinarily complex chemical system adopt a useful molecular organization and, in this limited sense, the microemulsion chemistry resembles what happens in a living cell. Substantial practical issues remain: rates for a recalcitrant VX simulant should be increased and overoxidation of the m...

Solution conformation of model peptides with the use of particle beam LC/FT-IR spectrometry and electrospray mass spectrometry

Solution conformations of the peptides angiotensin I (Ang I) and POMC-X (an octapeptide fragment of proopiomelanocortin) were investigated with the use of particle beam liquid chromatography/Fourier transform infrared (LC/FT-IR) spectrometry. Gradient elution high-performance liquid chromatography (HPLC) with mobile phases that contain acetonitrile, 2-propanol, 0.1% heptafluorobutyric acid (HFBA) and 0.1% trifluoroacetic acid (TFA) were used. The conformations of both peptides were studied in 0.9% sodium chloride injection USP, 5% dextrose in water injection USP and sterile water for injection USP. Additional conformational studies over a pH range of 2–10, to a temperature of 75°C and after a storage time of 24 h were investigated. The studies indicated that the two peptides do not behave similarly under identical conditions. It was observed that both Ang I and POMC-X had slightly different conformations in the various parenteral solutions. It was also shown that the conformation of Ang I changed with both pH and temperature while POMC-X was conformationally stable to both temperature and pH. The identity of the peptides and the conformationally sensitive charge-state intensities of the peptides were investigated with electrospray ionization mass spectrometry (ESI/MS). The combination of infrared and mass spectrometric data allowed a thorough estimation of solution effects on the conformations of the model peptides.

Determination of Lipophilicity by Gradient Elution High-Performance Liquid Chromatography

A novel method for the determination of lipophilicity using a simple HPLC protocol based on gradient elution chromatography is presented and compared to the common isocratic log k'(w) procedure. Linear relationships with high correlation coefficients between both methods for biologically active nucleosides and cyclic nucleotides as well as for environmentally relevant aromatic hydrocarbons were found. A mathematical fit to support the empirically determined linear relationship is presented. It is shown that the observed relationship between log k'(w) and the apparent capacity factor (k'(g)) determined by gradient elution is derivable by theoretical considerations as well. Since the gradient method is much less time-consuming compared to other procedures, it represents a convenient alternative for determining lipophilicity data in the future.

High Performance Liquid Chromatography/Atmospheric Pressure Chemical Ionization‐Mass Spectrometry for the Determination of Carotenoids

The performance of electrospray ionization and atmospheric pressure chemical ionisation (APCI) for the analysis of carotenoids was investigated. On our instrument, APCI produced spectra which were readily interpreted, and the positive-ion mode proved applicable to all of the compounds. The separation of nine compounds in under 14 minutes by gradient elution high-performance liquid chromatography (HPLC) has been demonstrated. The performance characteristics of positive-ion HPLC/APCI-mass spectrometry have been investigated and documented for the determination of nine carotenoids. With the exception of β-carotene, the precision of the method expressed as coefficient of variation ranged from 6 to 20%. Limits of detection ranged from 0.1 to 1.4 ng/μL dependent upon the analyte. The method was used to confirm the levels of carotenoids in UK Total Diet Survey samples.

High Performance Liquid Chromatography/Atmospheric Pressure Chemical Ionization-Mass Spectrometry for the Determination of Carotenoids

The performance of electrospray ionization and atmospheric pressure chemical ionisation (APCI) for the analysis of carotenoids was investigated. On our instrument, APCI produced spectra which were readily interpreted, and the positive-ion mode proved applicable to all of the compounds. The separation of nine compounds in under 14 minutes by gradient elution high-performance liquid chromatography (HPLC) has been demonstrated. The performance characteristics of positive-ion HPLC/APCI-mass spectrometry have been investigated and documented for the determination of nine carotenoids. With the exception of β-carotene, the precision of the method expressed as coefficient of variation ranged from 6 to 20%. Limits of detection ranged from 0.1 to 1.4 ng/μL dependent upon the analyte. The method was used to confirm the levels of carotenoids in UK Total Diet Survey samples.

Distribution and elimination of [ 14C] sarafloxacin hydrochloride from tissues of juvenile channel catfish (Ictalurus punctatus)

The distribution and loss of radioactivity from tissues were determined in 60 juvenile channel catfish ( Ictalurus punctatus) following oral dosing with the candidate fish therapeutant Sarafin ® ([ 14C] sarafloxacin hydrochloride) at 10 mg/kg for 5 consecutive days. Twelve groups of 5 fish each were sampled at selected times ranging from 3 to 240 h after the last dose was administered. The concentration and content of sarafloxacin-equivalent activity was determined in liver, gallbladder, kidney, skin, and skinless fillet by sample oxidation and liquid scintillation counting; content of sarafloxacin-equivalent activity was determined in stomach and anterior and posterior intestines. Skinless fillet tissues were also analyzed for sarafloxacin and for potential metabolites by gradientelution high-performance liquid chromatography (HPLC) with in-line radiometric and fluorescence detection. Loss of radioactivity from the whole body conformed to a bimodal elimination pattern with a rapid initial phase ( t 1 2 = 11 h ) and a slower secondary phase ( t 1 2 = 222 h ). Tissue and contents of the gastrointestinal tract (i.e. stomach and anterior and posterior intestines) were a principal depot of activity during the first four sample times (3, 6, 12, and 24 h); the combined head, skeleton, and fins (i.e. residual carcass) were the principal depot of activity in samples taken after 24 h. Of those tissues sampled 3 h after the last dose, relative sarafloxacin concentration was greatest in the liver (4.06 μg equivalents/g) and least in the residual carcass (1.13 μg equivalents/g). Intermediate concentrations were found in the kidney (2.04 μg equivalents/g), skinless fillet (1.71 μg equivalents/ g), and the skin (1.51 μg equivalents/g). Concentrations of sarafloxacin-equivalent residues in edible skinless fillet were consistently among the lowest of all tissues examined. The highest mean concentration of parent-equivalent material in the fillet tissue was found 12 h after administration of the last dose (2.27 μg equivalents/g) and declined thereafter. Sarafloxacin constituted between 80 and 90% of the extractable radioactive residues from the fillet homogenates. No other peaks were resolved in any of the fillet tissue samples analyzed by HPLC with in-line radiometric detection.

Compositional distribution characterization of poly(methyl methacrylate)-graft-polydimethylsiloxane copolymers

Graft copolymers prepared by radical polymerization of a low-molecular-mass monomer with a macromonomer display heterogeneity in both molecular mass and chemical composition. The characterization of these joint distributions by a single technique [e.g., size-exclusion chromatography (SEC)] is hindered by the effects of both variables on the separation mechanism. Separation emphasizing chemical composition heterogeneity can be efficiently performed by gradient elution high-performance liquid chromatography (HPLC) combining precipitation and adsorption retention. Comparison of Fourier transform IR and evaporative light-scattering detection indicated decreasing polydimethylsiloxane (PDMS) macromonomer incorporation corresponding to increasing retention time for a poly(methyl methacrylate) (PMMA)-graft-PDMS copolymer. More detailed information was obtained by multidetector SEC of composition fractions from gradient elution HPLC. SEC separation in an isorefractive solvent for PDMS (tetrahydrofuran) with low-angle laser-light scattering, differential viscometry, and differential refractive index detection allowed determination of the molecular mass of both the whole copolymer and that of the PMMA backbone for each HPLC fraction. Comparison with an independent SEC determination of the PDMS macromonomer molecular mass allowed estimation of the number of pendant PDMS chains per graft copolymer molecule across the HPLC chromatogram. Results indicated a relatively constant incorporation of the number of PDMS side chains with increasing PMMA backbone molecular mass, leading to a relative decrease in weight fraction PDMS incorporation with increasing molecular mass of the whole graft copolymer molecule.

Preparative capillary isotachophoresis as a sample pretreatment technique for complex ionic matrices in high-performance liquid chromatography

Preparative capillary isotachophoresis (ITP) was studied for sample pretreatment in the high-performance liquid chromatography (HPLC) of ionogenic analytes present in complex ionic matrices (urine and humic substances). Sulphanilate, methyl (4-aminobenzenesulphonyl)carbamate (asulam), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) served as model analytes. A high sample load of the ITP pretreatment was achieved by performing the preparative separations in tubes of 2.0 and 1.0 mm I.D. in the column-coupling configuration of the separation unit. The ITP separation according to ionic mobilities was combined with gradient elution HPLC in the ion-suppression mode to achieve highly dissimilar (orthogonal) separation systems in both techniques. The pretreatment provided the sample fraction for the HPLC analysis containing in addition to the analyte (sulphanilate) only ca. 2–3% of the urine matrix (spread along the complete elution profile) when a pair of discrete spacers defined the trapped constituents. Under these conditions the limit of detection for sulphanilate in urine could be reduced by more than two orders of magnitude. A high recovery of the pretreatment procedure [99 ± 1.5% for a 1.7 ppm (w/w) concentration of sulphanilate] was typical. For asulam, 2,4-D and MCPA present in a humic matrix it was shown that the ITP pretreatment may also be effective for multi-residue procedures while favourable analytical characteristics of the pretreatment such as recovery and efficient sample clean-up are maintained.

Separation of acid, basic and dispersed dyes by a single-gradient elution reversed-phase high-performance liquid chromatography system

A gradient elution high-performance liquid chromatography system with an end-capped narrow-bore reversed-phase column and photodiode-array detection has been developed for the separation and characterisation of acid, basic and dispersed dyes. The system exhibits good reproducibility, efficiency and sensitivity. Using a range of commercial dyes, a database of relative retention times and spectral data has been produced. The HPLC method has been applied to the analysis of dyes extracted from single fibres.

Separations of high-molecular-mass polystyrenes on different pore size and particle size reversed-phase columns in dichloromethane-acetonitrile

The effect of pore size and particle size of the silica support on the retention and resolution of high-molecular-mass polystyrenes was investigated in reversed-phase gradient elution high-performance liquid chromatography using a dichloromethane-acetonitrile mobile phase. An increase in pore size was found to increase retention slightly. A decrease in particle size was found to increase retention of all molecular masses. Both pore size and particle size had effects on resolution between polystyrenes of different molecular masses. Unretained polystyrene eluted at the solvent front for some of the conditions used but, this effect decreased with an increase in pore size and a decrease in particle size. A possible explanation for the greater effect on retention of particle size compared with pore size, is that the polystyrene molecules in a gradient elution experiment do not have access to the same number of pores as they do in an isocratic or size-exclusion experiment. It is proposed that this is due to a lag in the mobile phase composition in the pores compared with interstitial composition in the gradient experiment.