High-performance Liquid Chromatographic Method For Analysis Research Articles

High-performance liquid chromatography/tandem mass spectrometry for the quantitative analysis of a novel taxane derivative (BAY59-8862) in biological samples and characterisation of its metabolic profile in rat bile samples.

A sensitive, specific, accurate and reproducible high-performance liquid chromatography (HPLC) analytical method was developed and validated for the quantification of the novel oral taxane analogue BAY59-8862 in mouse plasma and tissue samples. A fully automated solid-phase extraction procedure was applied to the plasma after internal standard (IS) addition, with only 0.2 mL volume of the sample loaded on a CN-Sep-pak cartridge. In the case of the tissues a very simple acetonitrile extraction was used to recover BAY59-8862 and its internal standard from liver. The procedure for the quantification of BAY59-8862 and its IS (IDN5127) is based on high-performance liquid chromatography/ion spray-tandem mass spectrometry, operating in selected ion monitoring mode. The retention times of BAY and IS were 7.21 and 10.36 min, respectively. In both plasma and tissue specimens the assay was linear in the range 50-5000 ng/mL (ng/g). The overall precision and accuracy were assessed on three different days. The results for plasma were within 6.1% (precision) and between 99 and 112% (accuracy), and for the liver samples within 7.3% and between 104 and 118%, respectively. The LOD was 5 ng/mL and 20 ng/g in the plasma and liver, respectively. In addition, the biliary excretion of the compound in rats was studied. The study showed that an oxidative chemical reaction was the preferred metabolic pathway for biliary excretion, and two sets of mono- and dihydroxylated metabolites were detected by LC/ISP-MS/MS experiments. With this method, BAY59-8862 pharmacokinetic was determined in mice. The combined results demonstrate that the methodology can be considered a valid approach to conduct pharmacokinetic and metabolic studies during preclinical and clinical investigations.

A rapid high‐performance liquid chromatographic method for the determination of sinapine and sinapic acid in canola seed and meal

AbstractA high‐performance liquid chromatographic (HPLC) method has been developed to separate sinapine and sinapic acid from other phenolics in canola seed and meal in a single run. The separation was achieved with a reverse‐phase C18 column. Owing to the higher recovery of phenolics and ease of use, refluxing with 100% methanol for 20 min was selected as the extraction method for HPLC analysis and determination of total phenolics using Folin‐Ciocalteu reagent. A 10‐min isocratic/linear/concave gradient and a 15‐min isocratic/linear gradient were selected as the best gradients for the separation of these phenolic compounds. Peak identities for sinapine and sinapic acid were verified with ion exchange separation followed by HPLC analysis. The method was calibrated using sinapine bisulfate and sinapic acid standards; correlation coefficients (R2) for the calibration curves were 0.997 and 0.999 for sinapine bisulfate and sinapic acid, respectively. The extinction coefficient of sinapine was determined to be 1.16 times that of sinapic acid at the detector wavelength (330 nm). Applying this method to routine canola phenolic analyses can greatly reduce the cost by simplifying the procedures and reducing the time required for each determination.

High-performance liquid chromatographic determination of oxolinic acid and flumequine in the live fish feed Artemia

A high-performance liquid chromatography (HPLC) analytical method for the determination of oxolinic acid and flumequine in Artemia nauplii is described. The samples were extracted and cleaned up by a solid-phase extraction (SPE) procedure using SPE C 18 cartridges. Oxolinic acid and flumequine were determined by reversed-phase HPLC using a mobile phase of methanol–0.1 M phosphate buffer, pH 3 (45:55, v/v) and a UV detection wavelength of 254 nm. Calibration curves were linear for oxolinic acid in the range of 0.2–50 μg/g ( r 2=0.9998) and for flumequine in the range of 0.3–50 μg/g ( r 2=0.9994). Mean recoveries amounted to 100.8% and 98.4% for oxolinic acid and flumequine, respectively. The quantification limit was 0.2 μg/g for oxolinic acid and 0.3 μg/g for flumequine. Quantitative data from an in vivo feeding study indicated excellent uptake of both drugs by Artemia nauplii.

Rapid analysis of oxidized cholesterol derivatives by high‐performance liquid chromatography combined with diode‐array ultraviolet and evaporative laser light‐scattering detection

AbstractExtensive evidence of the deleterious biological effects of oxidized 5‐cholesten‐3β‐ol (cholesterol) derivatives has led to great interest in their detection. We observed that known oxidized cholesterol derivatives can be rapidly quantitated by combining reversed‐phase high‐performance liquid chromatography (HPLC) with ultraviolet (UV) absorption and evaporative laser light‐scattering (ELSD) detection. Using a 20 × 0.46 cm C18 HPLC column and methanol/acetonitrile (60:40, vol/vol) as the mobile phase at 1.0 mL/min, 10 species of oxidized cholesterol derivative were measured by UV (205, 234, and 280 nm) while 5‐cholestan‐5α,6α‐epoxy‐3β‐ol (5α‐epoxycholesterol), 5‐cholestan‐5β,6β‐epoxy‐3β‐ol (5β‐epoxycholesterol), and 5‐cholestan‐3β,5α,6β‐triol (cholestanetriol) were detected by only ELSD. The minimal limits of detection ranged from 100 to 500 ng depending on sterol and detector. The response was linear in the range 0–1000 or 0–2000 ng depending on detector. These oxidized cholesterol derivatives were also identified by HPLC/mass spectrometry analysis combined with UV detector. Heated tallow contained cholestanetriol, 5‐cholesten‐3β,7α‐diol (7α‐hydroxycholesterol), 5‐cholesten‐3β,7β‐diol (7β‐hydroxycholesterol), 5‐cholesten‐3β‐ol‐7‐one (7‐ketocholesterol), 5α‐ and 5β‐epoxycholesterols under the developed analysis condition. Photooxidized cholesterol had cholestanetriol, 7α‐ and 7β‐hydroxycholesterols and 3,5‐cholestadien‐7‐one. On the other hand, 7α‐ and 7β‐hydroxycholesterols, 7‐ketocholesterol, 5α‐ and 5β‐epoxycholesterols and 3,5‐cholestadien‐7‐one were observed in copper‐oxidized low‐density lipoprotein. Thus, this developed HPLC analysis method could be applied to identification of oxidized cholesterol derivatives in food and biological specimen.

Evaluation of the pharmacokinetic features and tissue distribution of the potent nonnucleoside inhibitor of HIV-1 reverse transcriptase, N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-240) with an analytical HPLC method.

The purpose of the present study was to examine the pharmacokinetic features and tissue distribution of N-[2-(2-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea (HI-240), a novel non-nucleoside inhibitor of HIV reverse transcriptase with potent anti-viral activity against AZT-sensitive as well as multidrug-resistant HIV-1 strains. A sensitive and accurate high performance liquid chromatography (HPLC)-based quantitative detection method was established to measure concentrations of HI-240 in pharmacokinetic studies. The plasma concentration-time data were modeled by using the WinNonlin program to estimate the pharmacokinetic parameter values. HI-240 had an elimination half-life of 78.3 +/- 2.0 min after i.v. administration and 196.8 +/- 3.1 min after i.p. administration. The systemic clearance of HI-240 was 2194 +/- 61 ml/h/kg after i.v. administration and 9339 +/- 1160 ml/h/kg after i.p. administration. Following i.v. injection, HI-240 rapidly distributed to and accumulated in multiple tissues with particularly high accumulation in adipose tissue, adrenal gland, and uterus+ovary. The concentration of HI-240 in brain tissue was comparable to that in the plasma, indicating that HI-240 easily crosses the blood-brain-barrier. Following i.p. injection, HI-240 was rapidly absorbed with a t1/2ka and a tmax values of less than 10 min. Following oral administration, HI-240 was absorbed with a t1/2ka of 4.2 +/- 1.1 min and a tmax of 95.1 +/- 25.1 min. The intraperitoneal bioavailability was estimated at 23.5%, while the oral bioavailability was only 1%. The HPLC-based accurate and precise analytical detection method and pilot pharmacokinetic studies described herein provide the basis for advanced preclinical pharmacodynamic studies of HI-240. The ability of HI-240 to distribute rapidly and extensively into extravascular compartments and easily cross the blood-brain barrier represent significant pharmacokinetic advantages over AZT.

High-performance liquid chromatographic method for the determination of nelfinavir, a novel HIV-1 protease inhibitor, in human plasma

Nelfinavir mesylate, a potent and orally bioavailable inhibitor of HIV-1 protease ( K i =2 n M), has undergone Phase III clinical evaluation in a large population of HIV-positive patients. A high-performance liquid chromatography analytical method was developed to determine the pharmacokinetic parameters of the free base, nelfinavir, in these human subjects. The method involved the extraction of nelfinavir and an internal standard, 6,7-dimethyl-2,3-di-(2-pyridyl)quinoxaline, from 250 μl of human plasma with a mixture of ethyl acetate–acetonitrile (90:10, v/v). The analysis was via ultraviolet detection at 220 nm using a reversed-phase C 18 analytical column and a mobile phase consisting of 25 m M monobasic sodium phosphate buffer (adjusted to pH 3.4 with phosphoric acid)–acetonitrile (58:42, v/v) that resolved the drug and internal standard peaks from non-specific substances in human plasma. The method was validated under Good Laboratory Practice (GLP) conditions for specificity, inter- and intra-assay precision and accuracy, absolute recovery and stability. The mean recovery ranged from 92.4 to 83.0% for nelfinavir and was 95.7% for the internal standard. The method was linear over a concentration range of 0.0300 μg/ml to 10 μg/ml, with a minimum quantifiable level of 0.0500 μg/ml for nelfinavir.

Determination of oxytetracycline in the live fish feed Artemia using high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic analytical method was developed for the determination of oxytetracycline in Artemia nauplii. A solid-phase extraction protocol was used to recover oxytetracycline and the internal standard tetracycline, from the Artemia samples. Oxytetracycline was analyzed using a 150 x 4.6 mm I.D. Hypersil-ODS column, a mobile phase of acetonitrile-tetrahydrofuran-0.01 M oxalic acid buffer (pH 3.0) (15:3:82, v/v), and an ultraviolet detection wavelength of 365 nm. The calibration curve of oxytetracycline in Artemia was linear (r2 = 0.9998) from 0.1 to 6.4 micrograms/g of tissue. Using a signal-to-noise ratio of 4:1 the oxytetracycline detection limit was 10 ng/g of tissue. Mean recovery of oxytetracycline amounted to 97%, while intra-assay variability was 1.5%. Quantitative data from an in-vivo feeding study indicated an excellent uptake of oxytetracycline by Artemia, as its levels reached 25.6 micrograms per g of nauplii.

Analyses of phenolic and furfural compounds in concentrated and non-concentrated apple juices

A high-performance liquid chromatography (HPLC) analytical method, using a combination of ultraviolet-diode array (UV) and electrochemical (EC) detectors, was developed for the characterization and quantification of phenolic and furfural compounds in apple juice. The limits of detection of phenolic compounds with EC detection were 4–500 times greater than those obtained with UV detection. The HPLC method allowed the identification and quantification of the major (chlorogenic and coumaroylquinic acids and phloridzin) and minor (caffeic, p-coumaric, ferulic, gallic and protocatechuic acids, and catechin) phenolic compounds, as well as 5-hydroxymethyl-2-furaldehyde and 2-furaldehyde in apple juice. The total phenolic content in apple juices of various origins varied from 28.85 to 115.49 mg/l. The concentrations of phenolic and furfural compounds varied widely among the non-concentrated and concentrated apple juices.

O 6-methylguanine-DNA adducts in rat lymphocytes after in vivo exposure to N-nitrosodimethylamine (NDMA)

A non-invasive approach in immunopathological risk assessment was applied for analysis of the in vivo formation of DNA adducts. DNA methylation was studied in peripheral blood lymphocytes (PBLs) collected from Sprague-Dawley rats exposed to a single dose (75 mg/kg b.w.) of N-nitrosodimethylamine (NDMA). Three different techniques were applied for characterization and quantification of DNA adducts: (i) colloidal gold ultraimmunocytochemical localization of O 6-methylguanosine (O 6-meG)-DNA adducts, using affinity-purified, polyclonal antibody directed against O 6-meG, (ii) quantitative assay using enzyme-linked immunosorbent assay (ELISA), amplified by the avidin-biotin (AB) system, and (iii) high-performance liquid chromatography (HPLC). The O 6-meG-immunoreactive sites in PBLs seem to be concentrated in the nucleus. However, significant immunolabelling was also noted in the cytoplasm of the in vivo NDMA-exposed PBLs. Control preparations showed no specific gold immunolabelling. The O 6-meG-DNA adduct formation in PBLs and hepatocytes, at 2–24 h following the exposure to NDMA, was analogous for both types of cells. The data showed high correlation for the ELISA and HPLC analytical methods. The data suggest an efficient O 6-metG-DNA repair mechanism in lymphocytes, possibly analogous to the enzymatic repair of DNA adducts in liver cells.

Direct high-performance liquid chromatographic separation of the racemates and diastereomers of nadolol

The direct separation of the two racemates and four diastereomers of nadolol by high-performance liquid chromatography (HPLC) is described. The racemates were well separated on non-chiral ODS columns using mixtures of phosphate buffer and methanol. The diastereomers could be separated on commercial chiral columns, using organic and aqueous mobile phases. The analytical HPLC method using an organic mobile phase was applied to semi-preparative HPLC for diastereomers.

Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography

A procedure employing C 18 reversed-phase high-performance liquid chromatography (HPLC) is described for evaluating the kinetics of biotinylation of specific residues of peptides after reaction with N-hydroxysuccinimide esters of biotin. Utilizing this HPLC method we determined the observed pseudo-first-order reaction rate constant ( k′ 1) of biotinylation of lysyl residues in two model peptides [biotinyl-Ser 108]ProA-egg laying hormone (108-121) and pGlu-Lys-Trp-Ala-Pro, to be 1.22 · 10 −2 s −1 and 1.08 · 10 −2 s −1, respectively in 0.05 M sodium phosphate buffer, pH 8.2, at 25°C. The respective reaction half-lives of the two peptides were 57 s and 64 s. In addition, HPLC analytical methods were established for determining the time-course of hydrolysis of biotinylating reagents at acidic alkaline pH and for evaluating biotin reagent homogeneity.

Homogentisic acid determined in biological fluids by HPLC.

In this rapid, specific, and sensitive high-performance liquid-chromatographic method of analysis for homogentisic acid in biological fluids, homogentisic acid is separated on a column of Nucleosil CN. This method, which we applied to the diagnosis of three cases of alcaptonuria, represents a suitable analytical tool for the diagnosis of alcaptonuria.

Improved method for HPLC analysis of polyamines, agmatine and aromatic monoamines in plant tissue.

The high performance liquid chromatographic (HPLC) method of Flores and Galston (1982 Plant Physiol 69: 701) for the separation and quantitation of benzoylated polyamines in plant tissues has been widely adopted by other workers. However, due to previously unrecognized problems associated with the derivatization of agmatine, this important intermediate in plant polyamine metabolism cannot be quantitated using this method. Also, two polyamines, putrescine and diaminopropane, also are not well resolved using this method. A simple modification of the original HPLC procedure greatly improves the separation and quantitation of these amines, and further allows the simulation analysis of phenethylamine and tyramine, which are major monoamine constituents of tobacco and other plant tissues. We have used this modified HPLC method to characterize amine titers in suspension cultured carrot (Daucas carota L.) cells and tobacco (Nicotiana tabacum L.) leaf tissues.

Isocratic Separation of Phenytoin Metabolites by High-Performance Liquid Chromatography from Human and Animal Microsomes and Urine

A rapid and simple high-performance liquid chromatography analytical method is described for the quantitative determination of phenytoin and five of its metabolites--phenytoin dihydrodiol, catechol, methoxycatechol, para-hydroxyphenytoin, and meta-hydroxyphenytoin--in biological materials. Following ethyl acetate extraction and incubation with beta-glucuronidase, samples were passed through a C18 Sep-Pak cartridge. The eluate was chromatographed on a reverse-phase 10 cm C18 Radial Nova-Pak (5 micron) column using a mobile phase of isopropanol:water (20:80) at a flow rate of 1.4 ml/min and monitored at 235 nm. Total chromatographic analysis time was 23 min, with complete baseline resolution of all metabolites. Five different columns and 10 different mobile phase conditions were studied to determine the best system. The 10-cm C18 Radial Nova-Pak (5 micron) gave the best resolution, reproducibility, and durability. This method should prove applicable for clinical use as well as for research purposes.

High-performance liquid chromatographic evaluation of salicyloyl pyridixol and systemic metabolites in biological samples

An high-performance liquid chromatographic analytical method, which allows quantitative evaluation of both salicyloyl pyridixol and its metabolite salicylic acid, is reported. This method has demonstrated to possess the required specifications in terms of linearity, sensitivity, extraction recovery, reproducibility and specificity for pharmacokinetic investigations in both human subjects and experimental animals. The results obtained from an investigation on the rat are briefly discussed.

Pregnenolone production by adrenal mitochondria: a new high-performance liquid chromatographic analytical method for cholesterol side-chain cleavage

Pregnenolone production by adrenal mitochondria: a new high-performance liquid chromatographic analytical method for cholesterol side-chain cleavage

Determination of Antimicrobial Preservatives in Pharmaceutical Formulations Using Reverse-Phase Liquid Chromatography

□ A specific stability-indicating reverse-phase high-performance liquid chromatographic analytical method has been developed to quantitate the antimicrobial preservatives methylparaben, propylparaben, butylparaben, sorbic acid, and benzoic acid in a series of typical pharmaceutical formulations. The mobile phase of this system is a water–acetonitrile mixture, modified by various acids and buffers. The proportions of water and acetonitrile as well as the type and amounts of modifiers are varied in order to achieve optimum chromatography. This method has been used successfully to quantitate preservatives in solutions, suspensions, creams, lotions, and ointments, and can be readily adapted to routine automated assays, either for routine product evaluation or stability programs.