Isocratic High-performance Liquid Chromatographic System Research Articles

Method for the Determination of Lycopene in Supplements and Raw Material by Reversed-Phase Liquid Chromatography: Single-Laboratory Validation

A single laboratory validation (SLV) study was conducted for a liquid chromatography (LC) method for the determination of total and all-trans-beta-carotene in a variety of dietary supplements, including multivitamin tablets, softgels, capsules, and beadlet raw materials. Extraction variants were developed for the different types of supplements tested based upon the supplement type and level of beta-carotene. Water dispersible formulations such as powders, emulsions, tablets, and capsules were enzymatically digested with protease and extracted with dichloromethane-ethanol. Oily suspensions were directly dissolved in dichloromethane-ethanol. After appropriate dilution or concentration, the extracts were chromatographed by using either a reversed-phase C18 column or, in products containing high amounts of alpha-carotene, a reversed-phase C30 column. The LC systems provided linear responses in the range of 0.1-50 microg beta-carotene/mL. The main geometrical isomers of beta-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were well separated from each other and from other carotenoids such as a-carotene, cryptoxanthin, lutein, lycopene, and zeaxanthin. Duplicate determinations of total beta-carotene performed by 2 technicians in 8 different test materials on 5 different days resulted in relative standard deviations of 1.2-4.4%. Recoveries determined for supplements and beadlet raw material spiked with beta-carotene levels of 10 microg to 100 mg/test portion and 0.2-40%, respectively, ranged from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results from the SLV study, the method is suggested for a collaborative study on the determination of total and all-trans-beta-carotene in dietary supplements.

High performance liquid chromatographic determination of N-butyryl glucosamine in rat plasma

Purpose: A high performance liquid chromatography (HPLC) method for determination in plasma of N-butyryl glucosamine (GLBU), a highly water-soluble compound with no chromophore was developed. Method: To 100 μL of plasma containing GLBU was added fucose as internal standard. GLBU and fucose were derivatized using 1-phenyl-3-methyl-5-pyrazolone in the presence of sodium hydroxide at 70 °C for 30 min. The solution was neutralized with hydrochloric acid and the excess derivatizing reagent was extracted with chloroform. The aqueous layer was injected into an isocratic HPLC system consisting of an autoinjector, a single pump and a UV detector set at 245 nm. Two different 25 cm reversed phase columns were used, a 4 and a 10 μm C 18 columns. The mobile phase was a mixture of phosphate buffer (pH 7) and acetonitrile (80:20), which was run through a pump at a flow rate of 1.0 mL/min at ambient temperature. Results: Derivatized fucose and GLBU appeared 24 and 28 min, and at 34 and 37 min using 4 and 10 μm columns, respectively. The assay was linear over the range of 0.2–200 μg/mL with a limit of quantification of 0.2 and 1 μg/mL for the 4 and 10 μm columns, respectively. The method was applied to the determination of GLBU in rat plasma after oral administration of 233 mg/kg of GLBU. Conclusion: The present assay is precise, and accurate with sufficient sensitivity for pharmacokinetic studies following therapeutically relevant doses.

Residues of ochratoxin A in pet foods, canine and feline kidneys.

The occurrence of ochratoxin A (OTA) in canned (26 samples) as well as dry pet foods (17 samples) for cats and dogs was investigated. In addition, 26 feline kidney samples with or without kidney alterations were surveyed for OTA-residues. The separation and detection of OTA was carried out by an isocratic high-performance liquid chromatography system based on reversed phase with fluorescence detection. After homogenization and extraction steps, immuno-affinity columns were applied for sample clean up. OTA could be detected in 47% (n=40) of the pet food samples. Those found positive contained generally low amounts of OTA (0.1-0.8 microg/kg original substance). Higher levels were only detected in two pet food samples (3.2 and 13.1 microg/kg toxin, respectively). Low concentrations of ochratoxin A could also be found iIn tissue of cat kidneys, with 16 of the analysed kidneys (n=26) being positive. The concentration levels were between 0.35 and 1.5 microg/kg OTA in tissue. No relation between pathological findings and ochratoxin levels in feline kidneys could be assessed.

Detection, Quantitation, and Identification of Residual Aminopenicillins by High-Performance Liquid Chromatography after Fluorescamine Derivation

A high-performance liquid chromatographic (HPLC) method with fluorescence detection after precolumn fluorescamine derivation was developed to detect residues of two aminopenicillins, amoxicillin (AMPC) and ampicillin (ABPC), in bovine serum. Proteins in serum samples spiked with each of these penicillins were precipitated with sodium tungstate and sulfuric acid, centrifuged, and removed by passage through a C18 solid-phase extraction cartridge. After precolumn treatment of the extraction products of AMPC and ABPC with fluorescamine solution, HPLC analysis with fluorescence spectrophotometric detection at an excitation wavelength of 390nm and an emission wavelength of 485nm was performed to identify these products. Two mobile phases were used for residual analysis by the isocratic HPLC system. An ODP column (polyvinyl alcohol bonded with an octadecyl functional group) that can be used with strongly alkaline mobile phases (pH 2.0 to 13) was selected, and the column temperature was set at 40°C. A mobile phase comprising 100-mM K2HPO4 solution and acetonitrile (72:28, vol/vol), which yielded AMPC and ABPC retention times of 4.1 and 7.9 min, respectively, was suitable for detection of residual ABPC but not for residual AMPC because interference was caused by peaks of other extracted substances. When a mobile phase comprising a different ratio of 100-mM K2HPO4 solution and acetonitrile (78:22, vol/vol) was used, the retention times of AMPC and ABPC were 7.3 and 26.3 min, respectively, and both penicillins could be analyzed using this system. The calculated standard curves of the reaction products with both mobile phases were linear, and the correlation coefficients were greater than 0.999. The lower limit of detection was 10 ng/ml for both penicillins. Analysis of extracts from bovine serum spiked with AMPC and ABPC at a concentration of 1 μg/ml yielded recovery rates of 102.2 ± 5.5% and 79.0 ± 5.2%, respectively. This detection method may be useful for routine laboratory testing of AMPC and ABPC.

Simple method for determination of terbutaline plasma concentration by high-performance liquid chromatography

A method is described in which low nanomolar concentrations of terbutaline in plasma can be quantitated by use of a standard isocratic high-performance liquid chromatography system with electrochemical detection. Samples were prepared for injection by solid-phase extraction and preserved from degradation by addition of glutathione. Terbutaline and internal standard metaproterenol were resolved from plasma constituents on a single C 18 column by ion-pairing chromatography. The method is precise and accurate for measurement of freebase concentrations as low as 4.4 nmol/l (1 ng/ml).

Biological monitoring of workers exposed to 4,4'-methylene-bis-(2-orthochloroaniline) (MOCA). I. A new and easy determination of "free" and "total" MOCA in urine.

The objective of the study was to validate a new and simple method to determine MOCA in the urine of exposed workers. The separation, identification and quantification of urinary MOCA were performed in spiked urines by a sensitive and practical high-performance liquid chromatography (HPLC) method and applied to urine samples of 11 workers occupationally exposed to MOCA; the postshift urinary levels of MOCA in their urine samples with and without hydrolysis, "total" and "free" MOCA respectively, were determined. In addition, we investigated the use of citric or sulfamic acid as preservatives of urine samples. The "total" and "free" MOCA were extracted with isooctane from hydrolysed and nonhydrolysed 20-ml urine samples respectively. After evaporation, the residue was dissolved in 4 ml of 2.10(-2) M aqueous hydrochloric acid and analysed by an isocratic HPLC system using both ultraviolet (UV) detection at 244 nm and electrochemical detection working in oxidation mode (0.9 V) with an Ag/AgCl reference electrode. Mobile phase (50% acetonitrile in water containing 0.4% acetate buffer solution pH = 4.6) was used to complete the 20-min analysis. "Free" and "total" MOCA were chromatographed on a reversed-phase C8 column (5 microns, 250 mm x 4 mm). The standard curve of MOCA was linear over the range 5-500 micrograms/l in human urine. The detection limit was 1 microgram/l for a 20-microliter injection volume; the repeatability ranged from 5.6 to 1.3% (n = 6) for spiked urines at 5 and 500 micrograms/l, with a percentage recovery of 94 +/- 3%. The reproducibility of the method was 7.3% (n = 4) for spiked urine at 10 micrograms/l. The use of sulfamic acid as a preservative of urine samples is important to improve the precision and accuracy of the analysis. The results indicate that these analytical procedures using conventional apparatus may be used routinely and reliably with large numbers of urine samples for biological monitoring of the exposure to MOCA. The occupational exposure to MOCA in some factories in France is studied in the second part of this work.

Quantification of the antipsychotics flupentixol and haloperidol in human serum by high-performance liquid chromatography with ultraviolet detection

A high-performance liquid chromatography (HPLC) method was developed for quantification of both isomers of the thioxanthene neuroleptic flupentixol and of the butyrophenone derivative haloperidol in human serum. After extraction with diethyl ether– n-heptane (50:50, v/v), an isocratic normal-phase HPLC system with a Hypersil cyanopropyl silica column (250×4.6 mm, 5 μm particle size) was used with ultraviolet detection at 254 nm and elution with a mixture of 920 ml acetonitrile, 110 ml methanol, 30 ml 0.1 M ammonium acetate, and 50 μl triethylamine. The limit of quantitation of 0.5 ng/ml and 0.3 ng/ml for flupentixol and haloperidol, respectively, was sufficient to quantify both compounds in serum after administration of clinically adjusted doses. The suitability of the described method for therapeutic drug monitoring and clinical pharmacokinetic studies was assessed by analysis of more than 100 trough level serum samples.

A Modified High Performance Liquid Chromatographic Analysis of Thiabendazole in Fruits and Vegetables with Elisa Confirmation

A rapid modified high performance liquid chromatographic (HPLC) method was developed for the analysis of the benzimidazole fungicide thiabendazole (TBZ) in fresh produce. Changes were in the extraction technique and HPLC mobile phase. TBZ was extracted from the produce by either polytroning 10 g in 20 mL of methanol for 3 minutes or shaking, by hand, a 5 or 10 g sample in 20 mL of methanol for 10 minutes. A 50 μL aliquot was injected directly into an isocratic fluorescent HPLC system. Total analysis time ranged from 8 to 15 minutes per sample depending upon the extraction procedure employed. Percent recoveries averaged 90% with excellent reproducibility (%CVs from 0 to 18). For enzyme linked immunosorbent assay (ELISA) confirmation a 0.1 mL aliquot of the sample was removed and evaporated to dryness under air. The residue was dissolved in water before performing the ELISA.

Method optimization and quality assurance in speciation analysis using high performance liquid chromatography with detection by inductively coupled plasma mass spectrometry

Achievement of optimum selectivity, sensitivity and robustness in speciation analysis using high performance liquid chromatography (HPLC) with inductively coupled mass spectrometry (ICP-MS) detection requires that each instrumental component is selected and optimized with a view to the ideal operating characteristics of the entire hyphenated system. An isocratic HPLC system, which employs an aqueous mobile phase with organic buffer constituents, is well suited for introduction into the ICP-MS because of the stability of the detector response and high degree of analyte sensitivity attained. Anion and cation exchange HPLC systems, which meet these requirements, were used for the seperation of selenium and arsenic species in crude extracts of biological samples. Furthermore, the signal-to-noise ratios obtained for these incompletely ionized elements in the argon ICP were further enhanced by a factor of four by continously introducing carbon as methanol via the mobile phase into the ICP. Sources of error in the HPLC system (column overload), in the sample introduction system (memory by organic solvents) and in the ICP-MS (spectroscopic interferences) and their prevention are also discussed. The optimized anion and cation exchange HPLC-ICP-MS systems were used for arsenic speciation in contaminated ground water and in an in-house shrimp reference sample. For the purpose of verification, HPLC coupled with tandem mass spectrometry with electrospray ionization was additionally used for arsenic speciation in the shrimp sample. With this analytical technique the HPLC retention time in combination with mass analysis of the molecular ions and their collision-induced fragments provide almost conclusive evidence of the identity of the analyte species. The speciation methods are validated by establishing a mass balance of the analytes in each fraction of the extraction procedure, by recovery of spikes and by employing and comparing independent techniques. The urgent need for reference materials certified for elemental species is stressed.

Risk of Neural Tube Defect-Affected Pregnancy Is Associated with a Block in Maternal One-Carbon Metabolism at the Level ofN-5-Methyltetrahydrofolate:Homocysteine Methyltransferase

The disposition of whole blood mono- to hexaglutamyl methylfolate and plasma homocysteine (HCY) was used to evaluate potential lesion sites in one-carbon metabolism which could be responsible for neural tube defect(NTD)-affected pregnancies. An isocratic high-performance liquid chromatographic system (HPLC) with photodiode array detection was used to quantify and speciate whole-blood methylfolate into mono-, di-, tri-, tetra-, penta-, and hexaglutamate forms. This technique was also used with off-line radioassay to identify nonmethyl whole-blood folates. Isocratic HPLC with fluorescence detection was used to quantify SBDF derivatized homocysteine in plasma. The study investigated blood from 11 women who had experienced a previous NTD- affected pregnancy and 11 controls of similar age and social class. No subjects were pregnant. HCY levels were significantly higher in NTD subjects (P= 0.0486, 95% CI −2.799,0.001 using the Mann–Whitney test), as was the ratio of known intracellular (tri- to hexaglutamyl) methylfolate compared to extracellular (mono- and diglutamyl) methylfolate (P= 0.0062 95% CI −0.543, 3.862 using the Mann–Whitney test). Vitamin B12, red cell folate, circulating total methylfolate, and circulating mono- to hexaglutamyl methylfolates showed no difference between population groups. The disposition between individual and cumulative glutamate chain lengths of methylfolate showed significant trends which differed between population groups: (i) total blood methylfolate (Glu1–6) appears to be utilized byN-5-methyltetrahydrofolate:homocysteine methyltransferase (MS) in control blood but not NTD blood, where it appears to accumulate following a 45-min incubation; (ii) whole-blood hexaglutamyl methylfolate (5CH3-H4PteGlu6) becomes a larger proportion of the total blood methylfolate in NTD than in control populations; and (iii) the intermediate glutamate chains of methylfolate (Glu1–5) remain relatively constant as 5CH3-H4PteGlu6accumulates in NTD but appear to increase linearly with 5CH3-H4PteGlu6in controls. The significant elevation of HCY in the NTD population is associated with the increasing proportion of 5CH3-H4PteGlu6relative to the total methylfolate, since, when corrected for HCY level, the proportion of 5CH3-H4PteGlu6to total methylfolate is similar in NTD and control populations. These trends are consistent with a defect at the level of vitamin B12dependent MS which “traps” folate at the 5CH3-H4PteGlu6level.

High-performance liquid chromatographic assay for the simultaneous determination of tramadol and its metabolites in microsomal fractions of human liver.

A high-performance liquid chromatographic assay for the quantitative determination of the opioid analgesic tramadol and its metabolites is described. A homologue of tramadol [1-(m-hydroxyphenyl)-2-(N-ethyl-N-methylaminomethyl)cycloheptane-1 -ol hydrochloride] is used as internal standard. The assay allows the determination of tramadol O- and N-demethylation activity in vitro in microsomal fractions of human liver. Tramadol and its in vitro generated Phase I metabolites are extracted by a one-step extraction procedure from microsomal incubation mixtures using methylene chloride. Extraction efficiencies of tramadol, O-demethyltramadol and mono-N-demethyltramadol were 70, 91 and 94% respectively. The isocratic high-performance liquid chromatographic system employs a C18 reversed-phase column. The mobile phase is a mixture of methanol, ammonium hydrogencarbonate solution and ammonium hydroxide solution. Sensitivity of the assay was 0.5, 0.2 and 0.2 microgram/ml for tramadol, O-demethyltramadol and mono-N-demethyltramadol, respectively. Within-run precision of the overall assay was 13, 3.1 and 7.6% for tramadol, O-demethyltramadol and mono-N-demethyltramadol, respectively. Accuracy of the assay was determined as mean differences of concentrations added and found in microsomal fractions. It was -2.4% for tramadol, -0.85% for O-demethyltramadol and 0.32% for mono-N-demethyltramadol.

Determination of the fungicides folpet, captan and captafol by cloud-point preconcentration and high-performance liquid chromatography with electrochemical detection

A method is described for the determination of residues of the fungicides Folpet, Captan and Captafol in river water using an isocratic high-performance liquid chromatographic system. Electrochemical detection with single and dual glassy-carbon electrodes was evaluated for possible amperometric detection of these fungicides; the reductive-oxidative detection mode with a dual electrode in the series configuration proved to be more appropriate than direct reductive detection with a single working electrode. To concentrate the fungicide residues, a cloud-point preconcentration step employing the non-ionic surfactant Triton X-114 was applied. Furthermore, the presence of the surfactant contributes to the stability of these analytes in aqueous solution, avoiding their hydrolysis. The method described shows good recoveries and good precision, with relative standard deviations of less than 10%. Detection limits of about 4 μg/1 were achieved for all three fungicides studied for 15 ml of preconcentrated sample.

Stepwise binary gradient high-performance liquid chromatographic system for routine drug monitoring

Drug therapy is usually optimized by concentration measurement in patient serum. High-performance liquid chromatography (HPLC) is one of the most important analytical techniques used for therapeutic drug monitoring (TDM) of drugs for which no immunoassay kits are available. HPLC has been frequently used for screening purposes in toxicology, too. The Merck Tox Screening System (MTSS) has been developed for the identification of substances by a combination of gradient HPLC with diode-array detection and identification with a database system. For routine TDM an isocratic HPLC system is more suitable because of shorter analysis time, better reproducibility of retention index and better precision of results. Therefore we defined a set of methods in steps of 10% of the two MTSS eluents. Three examples are shown: Amiodarone, Indometacine and Thiopental. New applications to test for other substances can be transferred to an isocratic system after a complete MTSS gradient run.

An isocratic high-performance liquid chromatographic system for simultaneous determination of theophylline and its major metabolites in human urine

A simple and highly selective isocratic high-performance liquid chromatography method is presented for the simultaneous determination of theophylline and its major metabolites in human urine using β-hydroxyethyl theophylline as an internal standard. The method utilizes direct injection of diluted urine samples followed by separation and quantitation by reversed-phase isocratic elution and ultraviolet detection. The assay is accurate and reproducible with a sensitivity of 1 μg ml −1 for theophylline and 0.5 μg ml −1 for its metabolites. The assay was employed for the analysis of theophylline and its major metabolites in urine following the oral administration of theophylline to four healthy volunteers.

Determination of semicarbazide-sensitive amine oxidase activity in human plasma by high-performance liquid chromatography with fluorimetric detection.

We report here a simple and sensitive method for the measurement of semicarbazide-sensitive amine oxidase (SSAO) activity in human plasma. Benzaldehyde, generated during a 1-h incubation of plasma with benzylamine, is derivatized with the specific aldehyde reagent dimedone after prior deproteinization. Quantitation of the derivatization product is done by automated injection onto an isocratic high-performance liquid chromatographic system with fluorimetric detection. The assay shows good linearity and reproducibility (intra-assay C.V. 7%). Detection limit is 25 mU/l (= pmol/ml/min). In 51 healthy controls (age 49 +/- 13 yr, 20 males) the measured SSAO activity was 352 +/- 102 mU/l (mean +/- S.D.). A large number of samples (70-80) can easily be processed in one day by one technician.

Determination of josamycin residues in porcine tissues using high-performance liquid chromatography with pre-column derivatization and spectrofluorimetric detection.

A simple, selective and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the measurement of josamycin residues in four porcine tissues (i.e., muscle, liver, kidney and fat). The sample preparation consisted of a homogenization step in an acetonitrile-10 mmol l-1 phosphate buffer mixture, pH 6.0 (35 + 65), centrifugation and a liquid-liquid extractive clean-up of the resulting supernatant with isooctane. Pre-column derivatization of josamycin was performed using cyclohexa-1,3-dione in ammonium acetate buffer, pH 5.0 (90 degrees C for 2 h). The derivative was chromatographed in an isocratic reversed-phase HPLC system. A LiChrospher RP 18 end-capped (5 microns) column was eluted with an acetonitrile-methanol-10 mmol l-1 phosphate buffer mixture, pH 6.0 (45 + 5 + 50). The capacity factor of the josamycin derivative was 17.5. Detection was achieved using spectrofluorimetry (lambda ex = 375 nm; lambda em = 450 nm). The structure of the derivative was assessed by using mass spectrometry. Full selectivity was obtained in the HPLC system versus other macrolide antibiotics (tylosin, spiramycin and erythromycin), aldehydes (formaldehyde, acetaldehyde and benzaldehyde) and endogenous compounds. Linearity and repeatability were tested. Correlation coefficients, for calibration curves in the range of 0.1-3.2 micrograms g-1, were greater than 0.999 for all tissues and the relative standard deviation (S(r)) was 4.9% (1.6 micrograms g-1; n = 6); recovery was higher than 88%.

Isolation and determination of AAL phytotoxins from corn cultures of the fungus Alternaria alternata f. sp. lycopersici

The fungus Alternaria alternata f. sp. lycopersici produces a group of four related host-specific phytotoxins (AAL toxins) which can be divided into two groups (TA and TB), each of which exists as an equilibrium mixture of two structural isomers. The AAL toxins were isolated from corn cultures by aqueous extraction, followed by purification on Amberlite XAD-2 resin, separation of TA from TB on silica gel and final purification on a semi-preparative high-performance liquid chromatographic (HPLC) system. A rapid, sensitive and reproducible method was developed to determine these toxins in culture material in order to monitor toxin production on corn cultures. The method consisted of aqueous extraction, C 18 solid-phase extraction clean-up, precolumn derivatization with o-phthaldialdehyde and reversed-phase HPLC with fluorescence detection. An isocratic HPLC system was developed that separated the structural isomers of TA and TB within a chromatographic analysis time of 24 min.

High-performance liquid chromatographic method for the simultaneous measurement of remacemide (a novel anticonvulsant and cerebroprotectant) and an active metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of both remacemide (a novel anticonvulsant and cerebroprotectant) and an active, major metabolite in human plasma. After the addition of an internal standard, the analytes were extracted from the plasma by ion-exchange solid-phase extraction and measured by an isocratic HPLC system with ultraviolet detection at 210 nm. The recovery of the analytes was >90%. The standard curves were linear over the range of quantitation of approximately 10–500 ng/ml for remacemide itself and 15–250 ng/ml for the metabolite. Both intra-day and inter-day accuracy and precision data were excellent. Remacemide and its metabolite were shown to be stable in human plasma for at least a year when stored at − 20°C.

Determination of Lauric Acid Metabolites in Peroxisome Proliferation After Derivatization and HPLC Analysis with Fluorimetric Detection

Abstract A derivatization procedure is described for the long chain fatty acid lauric acid and metabolites using the fluorescent probe 4-(bromomethyl)-7-methoxycoumarin. The derivatives can be separated in an isocratic high-performance liquid chromatographic system and detected using a fluorescence detector. The derivatization is rapid, simple and gives a good quantitation by the use of an internal standard. The procedure has been applied on samples rat liver homogenates from a toxicity experiment with the plasticizer di(2-ethylhexyl)phthalate. The activity of cytochrome P 450 IVA1 or lauric acid hydroxylase (LAH) in liver homogenates can be determined by the determina—tion of the 11- and 12-hydroxylated metabolites of lauric acid. The method turned out to be very sensitive and suitable to replace similar assays using radioactive compounds.

Determination of 6,7-Difluoro-1-(254-difluorophenyl)-1,4-dihydro-4-oxo-3-quinilinecarboxylic Acid and Related Compounds by High Performance Liquid Chromatography

Abstract 6,7-Difluoro- 1-(2,4-difluorophenyl)-1,4-dihydro-4-oxo-3-quinilinecarboxylic acid (Abbott-72931) and related compounds are determined by high performance liquid chromatography (HPLC). A two-part assay procedure quantitates both the drug substance and the related compounds. The chromatography used is based on a Bakerbond® C-4 column with a 0.04 M citrate buffer (pH 4.4) eluent modified with THF. Detection is at 254 nm. Quantitation of Abbott-72931 is achieved using an external standard method and an Abbott-72931 reference standard. An isocratic HPLC system is used with a 75% buffer/25% THF eluent. Assay precision (RSD values) for two lots of Abbott-72931 ranged from ± 0.66% to ± 0.97%. Quantitation of related compounds in Abbott-72931 is achieved by an external standard method using the same chromatography, with gradient elution to remove the more strongly retained impurities from the column. Unhydrolyzed Abbott-72931 ethyl ester is quantitated using an Abbott-72931 ethyl ester reference standard....