Liquid chromatographic method for the analysis of tocopherols in malt sprouts with supercritical fluid extraction.

A simple, specific and sensitive high-performance liquid chromatographic method has been developed for the determination of tocopherols in malt sprouts. A supercritical fluid extraction (SFE) procedure was used to isolate tocopherols from the vegetal matrix before quantitative analysis. The analytes were separated on a Zorbax reversed-phase column using methanol-water as mobile phase and quantified by measuring its fluorescence at lambda(em)=328 nm after excitation of the analytes at lambda(exc)=303 nm. The limits of detection for alpha-, gamma- and delta-tocopherols were 0.04, 0.05, and 0.05 microg/ml, respectively. The calibration graphs of the method were linear from 0.1 to 1.5, 0.2 to 2.5, and 0.2 to 2.0 microg/ml, for alpha-, gamma- and delta-tocopherols, respectively. This SFE and HPLC procedure is simple, precise and accurate for the determination of tocopherols in malt sprouts.     

HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).     

Determination of Heavy Polycyclic Aromatic Hydrocarbons (PAH) in Drinking Water,Ambient Air,Soils, Sediments and Food Stuffs by HPLC Method with Fluorescence Detection

The methods for the determination of some heavy PAH in different objects by HPLC method with fluorescence detection have been developed. The following conditions for the separation of PAH have been chosen:The column 150 mm length, internal diameter 2.1 mm filled by reversible-phase sorbent Zorbax ODS (5 μ),mobile phase acetonutrile/water (80:20), the injected volume of sample concentrate 10 μ L, the velocity of mobile phase 200 μ L.min. The excitation region is 290-300 nm, the emission one is 350-420 nm. Under condition chosen the limit of detection are 5 pg for benz(a)pyrene,10 pg for pyrene and chrysene3 pg for benz(k)fluoranthene, and 20 pg for anthracene. The dynamic range is 3 order of magnitude. The reproducibility of peak highs is better than 5%.     

Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid-phase extraction

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 μm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and β-carotene at 450 nm. Detection limits ranged from 0.2 μg/g (β-carotene) to 1.60 μg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale.     

An ion-pair reversed-phase HPLC-fluorimetric system for ultratrace determination of aluminium with salicylaldehydebenzoylhydrazone

An ion-pair HPLC fluorimetric determination of Al(III) at trace level has been developed, with salicylaldehydebenzoylhydrazone (SAB) as a precolumn reagent. The highly fluorescent AlSAB chelate (lambda(ex) 390.8 nm, lambda(em) 458.1 nm) is separated on a LiChroCART RP-18 column with an eluent consisting of 3.1 x 10(-)m tetrabutylammonium bromide, 1 x 10(4)m disodium EDTA and 5 x 10(-3)m sodium acetate in aqueous 42% w/w acetonitrile solution. The detection limit for Al is 1.5nM (40 pg/ml) in a 100-mul injection. The spectrophotometric detection limit at 390 nm is 0.3 ng/ml for 0.005 full-scale absorbance range. The selectivity is excellent and the method is useful for routine quality-control applications, such as determination of Al in tap water and in alkali pellets (LiOH, NaOH and KOH).     

Determination of vitamin E and carotenoid pigments by high performance liquid chromatography in shell of Chionoecetes opilio

This study reports the optimization of a method for the determination of vitamin E and carotenoids in shells of Chionoecetes opilio samples by online HPLC coupled with UV-vis and fluorescence detectors. The carotenoids were determined with diode-array detector (lambda 450 nm) and vitamin E with fluorescence detection (lambda(ex) 288, lambda(em) 331 nm). Two extractions methods were compared, saponification followed by an extraction step and a simple extraction with acetone. The last one was selected because allows to determine all compounds. Linearity, precisions and recoveries achieved for all compounds were satisfactory. Mean concentrations (mg per 100 g dry weight) were; 23.3 for vitamin E, 9.49 for astaxanthin and 0.2 mg for beta-carotene.     

Second Derivative Ultraviolet Spectrophotometry and High Performance Liquid Chromatography with Fluorometric Detection of Cycloserine Using 9-Chloro-10-methyl Acridinium Triflate as a New UV and Fluorescent Labeling Agent

Abstract

Sensitive and simple second derivative UV spectrophotometric and HPLC with fluorometric detection methods were developed for cycloserine based on derivatization with 9-chloro-10-methyl acridinium triflate (CMAT) to yield a reaction product which absorbs in the UV at 361 nm and is fluorescent using excitation and emission wavelengths of 257 nm and 475 nm, respectively. The CMAT derivatization reaction takes 30 minutes at 70°C. Cycloserine was linear in the 0.3 – 5.0 μg/ml range (r=0.999, n=5) for the second derivative UV method and the 0.8 – 5 μg/ml range for the HPLC method (r=0.999, n=5). The limit of detection for cycloserine in the HPLC method can be improved to 0.15 μg/ml with the addition of glacial acetic acid to the analytical sample. The HPLC assay was applied to the determination of cycloserine in spiked human urine samples. The correlation coefficient (r) was in the 0.999 range and sensitivity was at the low pg/ml level.     

Simultaneous determination of caffeine, theobromine, and theophylline by high-performance liquid chromatography

This work relates the development of an analytical methodology to simultaneously determine three methylxanthines (caffeine, theobromine, and theophylline) in beverages and urine samples based on reversed-phase high-performance liquid chromatography. Separation is made with a Bondesil C18 column using methanol-water-acetic acid or ethanol-water-acetic acid (20:75:5, v/v/v) as the mobile phase at 0.7 mL/min. Identification is made by absorbance detection at 273 nm. Under optimized conditions, the detection limit of the HPLC method is 0.1 pg/mL for all three methylxanthines. This method is applied to urine and to 25 different beverage samples, which included coffee, tea, chocolate, and coconut water. The concentration ranges determined in the beverages and urine are: < 0.1 pg/mL to 350 microg/mL and 3.21 microg/mL to 71.2 microg/mL for caffeine; < 0.1 pg/mL to 32 microg mL and < 0.1 pg/mL to 13.2 microg/mL for theobromine; < 0.1 pg/mL to 47 microg/mL and < 0.1 pg/mL to 66.3 microg/mL for theophylline. The method proposed in this study is rapid and suitable for the simultaneous quantitation of methylxanthines in beverages and human urine samples and requires no extraction step or derivatization.     

Determination of enrofloxacin and its primary metabolite, ciprofloxacin, in pig tissues. Application to residue studies

A simple and sensitive HPLC method has been developed for the simultaneous determination of enrofloxacin (ENR) and ciprofloxacin (CIP) in pig tissue using difloxacin (DIF) as internal standard. Tissue sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 m), followed by extraction with trichloromethane. Fluoroquinolones were separated on a reversed-phase column and eluted with aqueous buffer solution-acetonitrile (80:20, v/v). The concentrations of CIP, ENR and DIF eluted from the column, with retention times of 2.20, 2.73 and 4.38 min, respectively, were monitored by fluorescence detection at lambda(ex) 276 and lambda(em) 442 nm. The detection and quantitation limit were 8 and 25 ng/g, respectively, for both compounds. Standard curves were linearly related to concentration in the range 25-400 ng/g. The consequences of the introduction of minor reasonable variations (ruggedness studies) have also been analysed. Finally, the measurement of the tissue levels of ENR and CIP in the pig tissues after oral administration confirmed the utility of the proposed method.     

Sensitive determination of bisphenol A and alkylphenols by high performance liquid chromatography with pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole

A new and sensitive high-performance chromatographic method for the determination of bisphenol A and 8 alkylphenols with fluorescence detection is reported. Each phenol was derivatized by reaction with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole at 40 degrees C for 60 min. The fluorescence derivatives were separated on a Wakosil 5C18 column (4.0 i.d. x 300 mm, 5 microm) with methanol:water (10:90) as mobile phase (detection wavelength: lambda(ex) 336 nm, lambda(em) 440 nm). The detection limits were in the range of 0.1-10.0 pg/mL in serum. The calibration graphs were linear to 1.0 microg/mL. The relative standard deviations were 7.2-8.9%, respectively. The proposed method was applied to the determination of bisphenol A in mother and infant rat serum.     

High-Performance Liquid Chromatographic Determination of Progesterone

Abstract

A practical, rapid, reliable and isocratic reversed-phase HPLC method is described for the determination of progesterone; a method important for determining in cows (1) early pregnancy, (2) reproductive disorders, and (3) timing of artificial insemination. The method is reproducible with a detection limit of 0.5 ng/peak (400 pg/μl) at 254 nm (0.005 AUFS); down by nearly 10-fold from other methods and using simple 254 nm detection. Radioimmunoassay of all eluting fractions demonstrated specificity.     

Simultaneous determination of retinol and tocopherols by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method to determine retinol and all four tocopherols (alpha-, beta-, gamma- and delta-) simultaneously was established using a reversed-phase column (YMC-PACK A-302 S-5 120A ODS). The HPLC conditions were mobile phase 65% isopropanol, sample solvent 99.5% methanol and temperature 30 degrees C. Retinol and tocopherols were measured in rat liver.     

Direct fluorescence detection of Pb2+ and Cd2+ by high-performance liquid chromatography using 1-(4-aminobenzyl)ethylenediamine-N,N,N',N'-tetraacetate as a pre-column derivatizing agent

A highly sensitive HPLC with direct fluorescent detection (lambda(ex) = 235 nm, lambda(em) = 355 nm) was developed for Pb2+ and Cd2+ complexes with an aromatic polyaminocarboxylate, 1-(4-aminobenzyl)ethylenediamine-N,N,N',N'-tetraacetate as a pre-column derivatizing agent. A reversed phase partition column pretreated by a cationic surfactant was employed. Although this ligand forms thermodynamically stable complexes with various metal ions, only peaks of Pb2+ and Cd2+ were detected with the ligand-centered emission in the HPLC due to the emissive activity and kinetic stability in the dissociation reaction. The detection limits obtained were 1.5x10(-8) and 3.3x10(-9) mol l(-1) for Pb2+ and Cd2+, respectively.     

High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenylmethylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-micron, 250 X 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile-water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile-methanol-tetrahydrofuran-water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (lambda max = 264) was about 5 ng/ml and by fluorescence detection (lambda excitation = 270 nm, lambda emission = 300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 +/- 4.2% from serum and 102.0 +/- 2.62% from urine.     

Sample preparation for routine high-performance liquid chromatographic determination of retinol palmitate in emulsified nutritional supplements by solid-phase extraction using monosodium L-glutamate as dissolving agent

Retinol palmitate (vitamin A, 73.3 microg/g) in an emulsified nutritional supplement was determined by solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with fluorescence detection (excitation 350 nm, emission 480 nm) using monosodium L-glutamate as a dissolving agent to obtain higher recovery of vitamin A from the emulsified sample solution. A Bond Elut C2 cartridge (500 mg) was chosen for SPE after comparison with 16 other types. A sample solution was applied to a conditioned Bond Elut C2 cartridge and then vitamin A was eluted with ethanol followed by HPLC. The proposed method was simple, rapid (sample preparation time by SPE: ca. 8 min, retention time: ca. 8 min), sensitive [detection limit: ca. 0.1 pg/injection (100 microl) at a signal-to-noise ratio of 3:1], highly selective and reproducible (relative standard deviation (RSD): ca. 2.9% (n = 5), between-day RSD ca. 3.7 (5 days). The recovery of vitamin A was over 90% by the standard addition method.     

Rapid, sensitive and fully automated high-performance liquid chromatographic assay with fluorescence detection for sulmazole and metabolites

Sulmazole (2-[(2-methoxy-4-methylsulfinyl)phenyl]-3H-imidazo [4,5-b] pyridine; AR-L 115 BS) and two metabolites (sulfide, sulfone) were quantified from directly injected body fluids (plasma, urine, bile) after high-performance liquid chromatographic separation. No internal standard is needed, which is particularly advantageous when fluorescence detection is established. After automated pre-column enrichment on Corasil C18 (37-50 microns), the parent compound and biotransformation products could be backflushed and chromatographed on ODS-Hypersil (5 microns) with a mixture of 0.075 mol/l phosphate buffer-acetonitrile (2:1), an elution rate of 2.0 ml/min and fluorimetric detection (lambda ex = 330 nm; lambda em = 370 nm). A hydroxylated metabolite of sulmazole which occurs preferentially in urine (and bile) can be quantified in the above-mentioned solvent system diluted 1:1 with water, but with different fluorescence characteristics (lambda ex = 345 nm; lambda em = 515 nm). The assay was linear in the range 8-1000 ng/ml. The lower limit of detection was about 8 ng/ml or 80 pg with coefficients of variation between 0.4 and 5.8% for sulmazole.     

Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection

N-acetyl-l-cysteine (NAC) is a well-known antioxidant that is capable of facilitating glutathione (GSH) biosynthesis and replenishing intracellular GSH under oxidatively challenging circumstances. N-acetyl-cysteine-amide (NACA), the amide form of NAC, is a newly designed and synthesized thiol-containing compound which is believed to be more lipophilic and permeable through cell membranes than NAC. The metabolic and antioxidant effects of these compounds in vitro and in vivo are under investigation. However, an analytical method that can separate and quantify both compounds simultaneously is not yet available, to the best of our knowledge. Because of their structural similarities, the two compounds are difficult to separate using earlier HPLC methods which were designed for NAC quantification. Therefore, the goal of this work was to develop an HPLC method with fluorescence detection for simultaneous quantification of NAC and NACA in biological blood and tissue samples. A gradient HPLC program with fluorescence detection (lambda(ex) = 330 nm, lambda(em) = 376 nm) using N-(1-pyrenyl)maleimide (NPM) as the derivatizing agent was developed. The calibration curves were linear over a concentration range of 25-5000 nm (r(2) > 0.997). The coefficients of variation for within-run precision and between-run precision ranged from 0.67 to 5.23% and for accuracy ranged from 0.98 to 10.54%; the percentage relative recovery ranged from 94.5 to 102.8%. This new method provides satisfactory separation of NAC and NACA, along with other biological thiols, in 20 min with a 5 nm limit of detection (LOD) per 5 microL injection volume.     

High-performance liquid chromatography with fluorescence detection for the simultaneous determination of 3,4-methylenedioxymethamphetamine, methamphetamine and their metabolites in human hair using DIB-Cl as a label

This paper describes a highly sensitive HPLC method for the simultaneous determination of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in human hair samples. The amphetamines investigated were derivatized with the fluorescent reagent, DIB-Cl to yield highly fluorescent DIB-derivatives, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 nm and 430 nm, respectively. The separation was achieved on an ODS column with an isocratic mobile phase composed of acetonitrile-methanol-water (30:40:30, v/v/v). The limits of detection for the four compounds obtained by the proposed method ranged from 11 to 200 pg/mg. The method was successfully applied to the determination of MDMA and MDA in hair samples obtained from MDMA abuser.     

HPLC-UV method development and validation for the determination of low level formaldehyde in a drug substance

A reversed-phase high-performance liquid chromatographic method (HPLC) with diode-array detection is developed and validated for the quantitative determination of formaldehyde in a drug substance. Formaldehyde (HCHO) is reacted with 2,4-dinitrophenylhydrazine (DNPH) to form a Schiff base (HCHO-DNPH derivatization product), which has an absorbing maximum (lambda max) at 360 nm. The HPLC method employs a C8, 3-microm particle size analytical column (150 mm x 4.6 mm), 15-microL injection volume, column temperature controlled at 30 degrees C, detection at 360 nm, and a water-acetonitrile (55:45, v/v) mobile phase at a flow rate of 1 mL/min. These conditions resolve the HCHO-DNPH product from unreacted DNPH, the drug substance and related impurities, as well as diluent peaks within 20 min. The retention time of the HCHO-DNPH product is approximately 6.4 min. The method is linear, accurate in the specified range (0.33-333 ppm), and robust based on analyte (HCHO-DNPH derivatization product) stability in standard and sample. Detection limit is 0.03 ng (0.1 ppm).     

反相高效液相色谱法测定尿中吡啶醚和脱氧吡啶醚

尿中吡啶醚（ｐｙｒｉｄｉｎｏｌｉｎｅ,ＰＹＤ）和脱氧吡啶醚（ｄｅｏｘｙｐｙｒｉｄｉｎｏｌｉｎｅ,ＤＰＤ）是骨代谢特异的生化指标。应用高效液相色谱法（ＨＰＬＣ）建立了尿中ＰＹＤ和ＤＰＤ的测定方法。尿液用６ｍｏｌ／ＬＨＣｌ水解后,以纤维素ＣＦ１小柱提取,然后用ＨＰＬＣ测定;色谱系统为ＳｐｈｅｒｉｓｏｒｂＣ１８反相色谱柱,流动相组成为１５％甲醇添加０．１％七氟丁酸,流速为１．２ｍＬ／ｍｉｎ。系统的检测限：ＰＹＤ为１０ｎｍｏｌ／Ｌ,ＤＰＤ为７ｎｍｏｌ／Ｌ;回收率：ＰＹＤ为９１．５％,ＤＰＤ为１０６．１％;日内变异