Direct Injection High-performance Liquid Chromatography Research Articles

Rapid back flushed direct sample injection bio-analytical HPLC-UV method for therapeutic drug monitoring of terbinafine

A rapid back flushed (BF) direct sample injection (DSI) high-performance liquid chromatography (HPLC) with UV detection (BF-DSI-HPLC-UV) has been developed to determine terbinafine (TERB) in human serum. For online solid phase extraction step, an isocratic mobile phase of phosphate buffer saline (pH 7.4) at 1 mL/min and a short protein-coated ODS column (PC-ODS-column) were used for the purification and enrichment of TERB. Two different chromatographic modes of PC-ODS-column were simultaneously operated. Macromolecular proteins were extracted by size-exclusion liquid chromatography, while TERB trapping and enrichment were achieved through reversed-phase liquid chromatography. The clear fraction containing TERB was transferred from the PC-ODS-column by BF mode onto the quantification step through a high pressure switching valve. An analytical mobile phase consisting of 80% methanol and 1% triethylamine in distilled deionized water (pH) 6 at 1 mL/min was used for the final separation on an ODS analytical column. TERB was quantified and detected by UV-detector at 224 nm. The proposed method showed high correlation coefficient (>0.999) over the concentrations range 4–1600 ng/mL with recoveries ranging from 98.48 to 93.86%. Measurement of TERB concentration in serum after administration of a single dose of 250 mg oral tablet was used to evaluate the applicability of the BF-DSI-HPLC-UV for pharmacokinetic study.

Simultaneous determination of five common additives in insulating mineral oils by high-performance liquid chromatography with ultraviolet and coulometric detection.

Dielectric mineral oils are used to impregnate power transformers and large electrical apparatus, acting as both liquid insulation and heat dissipation media. Antioxidants and passivators are frequently added to mineral oils to enhance oxidation stability and reduce the electrostatic charging tendency, respectively. Since existing standard test methods only allow analysis of individual additives, new approaches are needed for the detection of mixtures. For the first time we investigate and discuss the performance of analytical methods, which require or do not require extraction as sample pretreatment, for the simultaneous reversed-phase high-performance liquid chromatography determination of passivators (benzotriazole, Irgamet(®) 39) and antioxidants (N-phenyl-1-naphtylamine, 2,6-di-tert-butylphenol, 2,6-di-tert-butyl-p-cresol), chosen for their presence in marketed oils. Quick easy, cheap, effective, rugged and safe and solid phase extractions were evaluated as sample pretreatments. Direct sample-injection was also studied. Ultraviolet spectrophotometry and direct-current coulometry detection were explored. As less prone to additive concentrations variability, the direct-injection high-performance liquid chromatography with ultraviolet and coulometric detection method was validated through comparison with Standard Method IEC 60666 and through an ASTM interlaboratory proficiency test. Obtained detection limits are (mg kg(-1) ): benzotriazole (2.8), Irgamet(®) 39 (13.8), N-phenyl-1-naphtylamine (11.9), 2,6-di-tert-butylphenol (13.1), 2,6-di-tert-butyl-p-cresol (10.2). Simultaneous determination of selected additives was possible both in unused and used oils, with good precision and accuracy.

Determination of rifampicin in rat plasma by modified large-volume direct injection RAM-HPLC and its application to a pharmcokinetic study.

A direct large volume injection high-performance liquid chromatography (HPLC) method with homemade restricted-access media (RAM) pre-column and combined with a column-switching valve was established and developed for determination rifampicin (RIP) in rat plasma. The rat plasma samples (100 μL) were injected directly onto pre-column, where RIP was retained and pre-concentrated, while proteins were washed to waste using a methanol-water (5:95) as the mobile phase at a flow rate of 1 mL/min. Then, by rotation of the switching valve at 5 min, the RIP were eluted from the pre-column and transferred to an Luna C18 analytical column by the chromatographic mobile phase consisting of methanol-acetonitrile-10 mm ammonium format (60:5:35) at a flow rate of 1 mL/min. The total analytical run time was 15 min with UV detection wavelength at 254 nm. Carbamazepine was used as the internal standard. Excellent linear correlation (r = 0.9993) was obtained in the range of 0.25-8 µg/mL for rat plasma. The intra-day and inter-day precisions of RIP were all <5.0%. The recoveries were in the range of from 99.98-113.66% for plasma. This on-line RAM-HPLC method was successfully applied to the pharmacokinetic study of RIP in rat plasma.

Analysis of Androgenic Steroids in Environmental Waters by Large-Volume Injection Liquid Chromatography Tandem Mass Spectrometry

A new method was developed for the analysis of natural and synthetic androgenic steroids and their selected metabolites in aquatic environmental matrixes using direct large-volume injection (LVI) high-performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). Method accuracy ranged from 87.6 to 108% for analytes with well-matched internal standards. Precision, quantified by relative standard deviation (RSD), was less than 12%. Detection limits for the method ranged from 1.2 to 360 ng/L. The method was demonstrated on a series of 1 h composite wastewater influent samples collected over a day with the purpose of assessing temporal profiles of androgen loads in wastewater. Testosterone, androstenedione, boldenone, and nandrolone were detected in the sample series at concentrations up to 290 ng/L and loads up to 535 mg/h. Boldenone, a synthetic androgen, had a temporal profile that was strongly correlated to testosterone, a natural human androgen, suggesting its source may be endogenous. An analysis of the sample particulate fraction revealed detectable amounts of sorbed testosterone and androstenedione. Androstenedione sorbed to the particulate fraction accounted for an estimated 5 to 7% of the total androstenedione mass.

Detection of s-triazine pesticides in natural waters by modified large-volume direct injection HPLC

There is a need for simple and inexpensive methods to quantify potentially harmful persistent pesticides often found in our water-ways and water distribution systems. This paper presents a simple, relatively inexpensive method for the detection of a group of commonly used pesticides (atrazine, simazine and hexazinone) in natural waters using large-volume direct injection high performance liquid chromatography (HPLC) utilizing a monolithic column and a single wavelength ultraviolet–visible light (UV–vis) detector. The best results for this system were obtained with a mobile phase made up of acetonitrile and water in a 30:70 ratio, a flow rate of 2.0 mL min −1, and a detector wavelength of 230 nm. Using this method, we achieved retention times of less than three minutes, and detection limits of 5.7 μg L −1 for atrazine, 4.7 μg L −1 for simazine and 4.0 μg L −1 for hexazinone. The performance of this method was validated with an inter-laboratory trial against a National Association of Testing Authorities (NATA) accredited liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) method commonly used in commercial laboratories.

Trans-resveratrol and Other Phenolic Compounds in Turkish Red Wines with HPLC

The moderate consumption of red wine has a relatively great benefit in the prevention of atherosclerosis and coronary heart disease (CDH). Resveratrol, quercetin, and other polyphenolic flavonoids constitute the phenol content of wine, which have also been reported to be cancer preventive agents. The grape varieties, climate, soil, the use of agronomic techniques, the health of grapes and wine making processes are the most important factors on the phenolic content of red wine. In this research, direct injection high-performance liquid chromatography (HPLC) is applied for the determination of trans-resveratrol and some phenolic antioxidants (catechin, epicatechin, quercetin and rutin) in sample wines. The wines are mostly known traditional Turkish red wines produced from different grape varieties originated from different geographical regions.

Quantitative determination of pioglitazone in human serum by direct-injection high-performance liquid chromatography mass spectrometry and its application to a bioequivalence study

A simple, high throughput, direct-injection high-performance liquid chromatography tandem mass spectrometry method (LC/MS/MS) has been developed and validated for the quantitation of pioglitazone in human serum. After mixing the internal standard with a sample, a 10 μl portion of the mixture was directly injected into a high-flow LC/MS/MS system, which included an extraction column, an analytical column and a six-port switching valve. The on-line extraction was achieved on an Oasis ® HLB column ( 1 mm×50 mm, 30 μm) with a 100% aqueous loading mobile phase containing 5 mM ammonium acetate (pH 4.0) at a flow rate of 4 ml/min. The extracted analyte was eluted by a mobile phase which contained 5 mM ammonium acetate and acetonitrile. The analytical column was a Luna C18 column ( 4.6 mm×50 mm, 5 μm). Detection was achieved by positive ion electrospray tandem mass spectrometry. The lower limit of quantitation of the method was 9 ng/ml. The standard curve, which ranged from 9 to 1350 ng/ml, was fitted by a weighted (1/ x 2) quadratic regression model. The validation results demonstrated that this method had satisfactory precision and accuracy across the calibration range. There was no evidence of instability of the analyte in human serum following three freeze-thaw cycles, and samples could be stored for at least 2 weeks at −30 °C. This method was used to analyze pioglitazone concentrations in human serum samples from a bioequivalence study of a blinded Actos ® formulation (encapsulated 15 mg tablet) and an Actos ® 15 mg tablet. The blinded formulation was shown to be bioequivalent to an Actos ® 15 mg tablet.

An on-line solid phase extraction—Liquid chromatography—Tandem mass spectrometry method for the analysis of citalopram, fluvoxamine, and paroxetine in human plasma

A specific and sensitive direct-injection high performance liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (HPLC-APCI-MS-MS) method has been developed for the rapid identification and quantitative determination of citalopram, fluvoxamine, and paroxetine in human plasma. After dilution with 0.1% formic acid, plasma samples were injected into the LC-MS-MS system. Proteins and other large biomolecules were removed during an on-line sample cleanup step. The inter- and intra-assay coefficients of variation for all compounds were <11%. The total analysis time was 6 min per sample. The proposed method permits direct analysis of plasma samples without time-consuming sample preparation.

Direct-injection high performance liquid chromatography ion trap mass spectrometry for the quantitative determination of olanzapine, clozapine and N-desmethylclozapine in human plasma.

A specific and sensitive direct-injection high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the rapid identification and quantitative determination of olanzapine, clozapine, and N-desmethylclozapine in human plasma. After the addition of the internal standard dibenzepin and dilution with 0.1% formic acid, plasma samples were injected into the LC/MS/MS system. Proteins and other large biomolecules were removed during an online sample cleanup using an extraction column (1 x 50 mm i.d., 30 microm) with a 100% aqueous mobile phase at a flow rate of 4 mL/min. The extraction column was subsequently brought inline with the analytical column by automatic valve switching. Analytes were separated on a 5 microm Symmetry C18 (Waters) analytical column (3.0 x 150 mm) with a mobile phase of acetonitrile/0.1% formic acid (20:80, v/v) at a flow rate of 0.5 mL/min. The total analysis time was 6 min per sample. The inter- and intra-assay coefficients of variation for all compounds were <11%. By eliminating the need for extensive sample preparation, the proposed method offers very large savings in total analysis time.

Direct HPLC Assay of Five Biologically Interesting Phenolic Antioxidants in Varietal Greek Red Wines

A convenient direct injection high-performance liquid chromatography (HPLC) method is introduced and applied to the simultaneous assay of five biologically interesting phenolic antioxidants (catechin, epicatechin, trans-resveratrol, quercetin and rutin) in the most characteristic Greek varietal red wines. Since the growing and winemaking procedures of the examined samples were identical, the observed quantitative differences are related to grape variety and geographical origin.

Determination of Hexazinone in Groundwater by Direct-Injection High-Performance Liquid Chromatography

Hexazinone has been detected at levels ranging from 0.2 to 50 micrograms/L in many groundwater samples from eastern Maine over the past decade. A rapid and inexpensive direct-injection high-performance liquid chromatographic (HPLC) method has been developed to monitor contamination levels of the herbicide. The method is sensitive (limit of quantitation = 0.33 microgram/L) and is linear to 33.0 micrograms/L (R2 = 0.9995). Direct injection results from 50 field samples compared well (R2 = 0.98) with an HPLC method using solid-phase extraction for concentration and cleanup. The technique is very reproducible (coefficients of variation of 0-8.4% within day and 3.0-13.2% between day) and eliminates loss of analyte because of fewer steps in the procedure.

Determination of a new oral cephalosporin, S-1090, in human plasma and urine by direct injection high-performance liquid chromatography with ultraviolet detection and column switching

Direct injection high-performance liquid chromatographic (HPLC) methods with column switching and UV detection were developed for the rapid and accurate determination of S-1090 in human plasma and urine. An internal-surface reversed-phase pre-column and a C 18 analytical column were used for the plasma assay. Two pre-columns packed with cyano and phenyl materials and a C 18 analytical column were used for the urine assay. The calibration curves for plasma and urine assays were linear in the ranges 0.09–9 μg/ml and 0.5–100 μg/ml of S-1090, respectively. The relative standard deviations for plasma and urine assays were less than 6% with low relative errors. The established HPLC methods were demonstrated to be useful for clinical pharmacokinetic studies after oral administration of S-1090.

Isocratic high-performance liquid chromatographic determination of thiacetazone by direct injection of plasma into an internal surface reversed-phase column

This report describes the determination of thiacetazone in human and rat plasma by direct-injection high-performance liquid chromatography (HPLC). Plasma filtrate (50 microliters) was injected directly into the internal surface reversed-phase (ISRP) mixed-functional phenyl column (Capcell Pak, 50 x 4.6 mm, 5 microns) and eluted with an aqueous mobile phase containing 7.5% acetonitrile at a flow-rate of 1 ml/min. With UV detection at 322 nm, thiacetazone eluted at 11.0 min whereas endogenous interferences eluted before 5 min. The lower detection limit for a 50-microliter sample at a signal-to-noise ratio of 5 was 63 ng/ml, which was several hundred fold lower than its cytotoxic concentrations determined from in vitro cell line studies. At a concentration range of 0.17 to 2.7 micrograms/ml, the recovery of thiacetazone was 98.0 +/- 4.4% (mean +/- S.D.). The intra- and inter-day coefficients of variation were 3.0 +/- 1.4% and 4.2 +/- 2.1%, respectively. This method was successfully applied to study the pharmacokinetics of thiacetazone in rats. The direct injection method is simple, requires small sample volume and does not require sample extraction, internal standard, or gradient elution.

Comparative Evaluation of Four Methods for Assay ofcis-andtrans-Resveratrol

A comparative evaluation of four methods to measure the concentrations of <i>cis-</i> and <i>trans-</i>resveratrol as well as total resveratrol in commercial wines has been performed. Two of these methods utilized solid-phase extraction of resveratrol isomers prior to analysis by direct-injection gas-chromatography mass-spectrometry (GC-MS) or derivatization with <i>bis</i>-[trimethylsilyl]-trifluoroacetamide (BSTFA) prior to GC-MS analysis. Two methods utilized direct-injection high performance liquid chromatography (HPLC) in normal phase (isocratic) with absorbance detection at 306 nm, and reverse phase HPLC with gradient elution and diode array detection. In virtually all comparisons, the correlation between the values for any two methods was very satisfactory (r &gt; 0.900), but some evidence of systematic bias was obtained which could not be explained by different standardization techniques. High values for <i>trans-</i>resveratrol with direct-injection GC-MS (Method 1) could be attributed, at least in part, to thermal breakdown of resveratrol glucosides to free isomers. The derivatization GC-MS technique (Method 2) showed a tendency to overestimate <i>cis-</i>resveratrol and underes- timate the trans isomer, possibly as a consequence of <i>trans-</i> to-<i>cis</i> isomerization during the derivatization step. Somewhat lower values for <i>cis-</i>resveratrol with the normal-phase HPLC procedure (Method 3) might be a consequence of monitoring a single wavelength (306 nm) which is well above the absorption maximum of this isomer. Method 4 (reverse-phase gradient HPLC with diode array detection) has the advantage of allowing the simultaneous quantitation of many other polyphenols of biologic interest, and as of now may be considered to be the most robust method for routine application.

Complementation of direct-injection high-performance liquid chromatography and enzyme-linked immunosorbent assay for the analysis of thiabendazole in fruit juices and concentrates

The fungicide thiabendazole was quantified in fruit juices and their concentrates (bulk and store bought) without clean-up by simply injecting 50 μl of dissolved sample into an HPLC. This novel method used a mobile phase consisting of acetonitrile-methanol-water-ethanolamine (37:11:52:0.02) and was passed through a C 18 column at 1 ml/min. Detection was accomplished by fluorescence at 305 nm excitation and 345 nm emission. Using peak height thiabendazole was linear from 0.032 to 2.05 ng injected. The limit of quantitation was 5 ppb for juices and store concentrates (2.0 ppb for the limit of detection), 10 ppb for bulk apple concentrates and 25 ppb for other bulk concentrates (5 and 10 ppb, respectively for the limit of detection). Intra- and interassay percent relative standard deviations for standards and samples were mostly below 7% with none above 9%. Eighty-four juices and concentrates out of 200 analyzed were found to contain thiabendazole (2–2560 ppb) by HPLC and all were shown to be thiabendazole positive by enzyme-linked immunosorbent assay (ELISA). The HPLC vs. ELISA correlation coefficient was 0.984.

Direct injection isocratic high-performance liquid chromatographic analysis of mitomycin C in plasma

A direct injection high-performance liquid chromatography method is described for the determination of mitomycin C (MMC) in human plasma. The stationary phase consisted of hydrophilic and hydrophobic functional groups covalently bound to silicone-coated silica beads (CAPCELL PAK MF Ph-1, 150×4.6 mm I.D., 5 μm). A mobile phase using 100% water gave a better separation of MMC from endogenous interferences as compared to a mobile phase with 12.5% acetonitrile and 2.5 m M phosphate buffer (pH 6.9). Using water as the eluent (1 ml/min) and UV detection at 365 nm, MMC was found to elute at 5.0 min with a peak width of 0.3 min, whereas endogenous interferences eluted before 3 min. Total assay time per sample was 6 min. Internal standard was not required because the recovery of MMC was nearly complete, about 90% from 20 to 5000 ng/ml. The standard curve was linear from 20 to 5000 ng/ml in plasma, and the intra- and inter-day variation was between 3 to 6%. The lower detection limit was 5 ng/ml with a 25 μl sample, which represent a two- to four-fold improvement over the 10 ng/ml detection limit by previous methods using liquid-liquid extraction and comparable sample size. The simplicity of this method, i.e., no sample extraction, no internal standard, 100% aqueous mobile phase, isocratic elution and short analysis time (6 min/sample), makes it suitable for large scale routine sample analysis, whereas its small sample volume requirement and high sensitivity are useful for pharmacokinetic studies in small animals where limited sample is available.

Assay of resveratrol glucosides and isomers in wine by direct-injection high-performance liquid chromatography

We have developed a normal-phase high-performance liquid chromatographic method to measure the glucosides of cis- and trans-resveratrol as well as the free isomers in commercial wines. The procedure utilises a novel technique for calibration requiring only the trans-resveratrol in pure form and the conversion of the glucosides to the free isomers by hydrolysis with β-glucosidase. Only 20 μl of sample is needed for direct injection without prior preparation, and isocratic elution separates the 4 compounds of interest in approximately 50 min. Calibration curves are linear, and stable for several months. Recovery was around 100% for all compounds, and replicate analyses gave coefficients of variation of 0.4–6.7%. The limits of sensitivity were 0.2 and 0.1 μmoles l −1 for the glucosides and free isomers of cis- and trans-resveratrol, respectively. A preliminary survey of sixteen wines from eight countries or regions revealed the presence of all four compounds in fourteen; the glucosides of resveratrol were present in higher concentrations than the free isomers in five of the wines.

IDENTIFICATION OF PROANTHOCYANIDINS IN BEER AND THEIR DIRECT MEASUREMENT WITH A DUAL ELECTRODE ELECTROCHEMICAL DETECTOR

A sensitive and reliable method was developed for the measurement of simple flavanols in beer by direct-injection High Performance Liquid Chromatography using dual-electrode electrochemical detection. When operated in the amperometric mode the detector was extremely sensitive for readily-oxidized substances such as polyphenols, so that sample preparation was minimal. The dimeric flavanols in a commercial beer were positively identified as procyanidin B3 and prodelphinidin B3. Tentative identifications for three trimeric proanthocyanidins were made also. Collectively, monomeric, dimeric and trimeric flavanols accounted for only about 4% of the colorimetrically determined total polyphenol content, and about 35% of the total flavanol content of a non-haze stabilized beer. The improved chromatographic method was applied to measuring the decreases in simple flavanols caused by treatment of beer with polyvinylpolypyrrolidone.

Analysis of neutral nitrogen compounds in diesel oil by direct injection high-performance liquid chromatography—mass spectrometry—ultraviolet spectrometry methods

An analytical methodology has been developed for the direct analysis of neutral nitrogen compounds in diesel oil by HPLC with subsequent detection using MS and/or photodiode-array UV spectrometry. By diluting diesel oils with one volume of hexane, diesel samples could be injected directly onto an alumina analytical column and then eluted with a gradient mobile phase consisting of hexane and dioxane. Nitrogen compounds were selectively retained on the alumina column, eluted using LC, and then detected by particle beam LC—MS or UV at 254, 270 and 290 nm. Two Brazilian and one Arabian diesel distillates were investigated. Alkylcarbazoles and alkylindoles were identified as the major neutral nitrogen components. Structural identification was accomplished by combination of electron impact MS and UV analyses. The concentration of carbazoles and indoles was estimated using calibration standards of carbazole and indole, respectively.

Rapid Determination of 4-Vinyl Guaiacol and Ferulic Acid in Beers and Worts by High-Performance Liquid Chromatography

Sensitive methods for the determination of ferulic acid and 4-vinyl guaiacol in beers and worts are described. Samples were analyzed by direct-injection high-performance liquid chromatography using an octadecyl silica column. Detection was by either fluorescence monitoring (for 4-vinyl guaiacol alone) or electrochemical detection (for simultaneous determination of ferulic acid and 4-vinyl guaiacol). The methods were applied mainly to the analysis of production beers but were used also to monitor the decarboxylation of ferulic acid to 4-vinyl guaiacol in wort during a small-scale fermentation deliberately contaminated with a wild yeast strain.