Narrow-bore High-performance Liquid Chromatography Research Articles

Fast Determination of Toxic Arsenic Species in Food Samples Using Narrow-bore High-Performance Liquid-Chromatography Inductively Coupled Plasma Mass Spectrometry.

A new method for the speciation analysis of arsenic in food using narrow-bore high-performance liquid-chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) has been developed. Fast separation of arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid was carried out in 7 min using an anion-exchange narrow-bore Nucleosil 100 SB column and 12 mM ammonium dihydrogen phosphate of pH 5.2 as the mobile phase, at a flow rate of 0.3 mL min(-1). A PFA-ST micronebulizer jointed to a cyclonic spray chamber was used for HPLC-ICP-MS coupling. Compared with standard-bore HPLC-ICP-MS, the new method has provided higher sensitivity, reduced mobile-phase consumption, a lower matrix plasma load and a shorter analysis time. The achieved instrumental limits of detection were in the 0.3 - 0.4 ng As mL(-1) range, and the precision was better than 3%. The arsenic compounds were efficiently (>80%) extracted from various food samples using a 1:5 methanol/water solution, with additional ultrasonic treatment for rice products. The applicability of this method was demonstrated by the analysis of several samples, such as seafood (fish, mussels, shrimps, edible algae) and rice-based products (Jasmine and Arborio rice, spaghetti, flour, crackers), including three certified reference materials.

Application of narrow-bore HPLC columns in rapid determination of sildenafil citrate in its pharmaceutical dosage forms.

A special type of silica-based columns has been recently introduced into the market which is called narrow-bore columns. They have lower internal volume than the standard high-performance liquid chromatography (HPLC) columns and thus reduce the solvent consumption by almost 80%. A simple, accurate and environmentally friendly reversed phase- HPLC (RP-HPLC method) which could be used in fast and high throughput analyses has been developed for the purpose of determining the sildenafil in bulk and pharmaceutical dosage forms, using narrow-bore C18 column (50 × 3.2 mm, 5 µm particle size) in isocratic mode, with mobile phase comprising of buffer (pH = 3) and acetonitrile in the ratio of 75:25 v/v. The flow rate was 0.7 mL/min and the detection was monitored through Ultraviolet detector (UV detector) at 292 nm. Clonazepam was used as the internal standard and the run time was 4 min. The proposed method has permitted the quantification of sildenafil over the linearity in the range of 30-4000 ng/mL and its percentage recovery was found to be 99-105%. Limit of quantitation (LOQ) is determined as 30 ng/mL. The intra-day and inter-day precisions were found 1.2-2.2% and 1.56-3.4% respectively. The solvent consumption was 2.8 mL per sample of which ca 0.7 mL was acetonitrile. This study shows that the application of narrow-bore column instead of the conventional reversed phase column in HPLC analyses has the advantages of shorter run time and less organic solvent consumption. This method is highly sensitive with excellent recoveries and precision and there is no need for special column and pre-column or post-column treatment of the sample. Moreover, the method is free from interference by common additives and excipients, suggesting applications in routine quality control analyses.

A validated method for the quantification of curcumin in plasma and brain tissue by fast narrow-bore high-performance liquid chromatography with fluorescence detection

Curcumin, a lipophilic polyphenol derived from the rhizome of the plant turmeric (Curcuma longa), might be useful in the prevention and treatment of a number of degenerative brain disorders, including glioma multiforma and Alzheimer's disease. Thus, there is growing interest in measuring curcumin concentrations in the brain and other target tissues in relevant animal models. We therefore developed and validated (according to the Food and Drug Administration guidelines for bioanalytical method validation), a simple, fast and reliable method for the quantification of curcumin in biological matrices by fast high-performance liquid chromatography with fluorescence detection. This method involves a simple extraction with 95% ethyl acetate and 5% methanol, rapid separation (<2 min if external standards and <4 min if the internal standard beta-estradiol 17-acetate is used) on a Jasco Reprosil-Pur Basic C(18) column (75 x 2 mm, 1.8 mum) with an eluent of acetonitrile, methanol, de-ionised water and acetic acid (49:20:30:1, v/v; flow rate, 0.4 mL/min) and fluorescence detection (excitation wavelength, 420 nm; emission wavelength, 470 nm). The method is selective, precise (<15% RSD at the lower limit of quantification), accurate (<15% of the coefficient of variation at the lower limit of quantification) and sensitive over a linear range of 0.05-10 microg/mL for curcumin. The developed method was used for the quantification of curcumin in the brains of mice force-fed (50 mg/kg bw) or i.p. injected (100 mg/kg bw) with curcumin. Brain curcumin concentrations of the mice were below the limit of detection at 30, 60 and 120 min after oral gavage and reached 4-5 microg/g brain 20-40 min after i.p. injection. In conclusion, the developed and validated method should be useful for the accurate and precise quantification of curcumin in target organs from relevant animal models of human diseases.

Down-scaling narrowbore LC-ICP-MS to capillary LC-ICP-MS: a comparative study of different introduction systems

Narrowbore high performance liquid chromatography (HPLC) was scaled down stepwise to capillary HPLC employing 2.1, 1 and 0.32 mm id separation columns. Provided that each column was operated at optimum chromatographic conditions (eluent velocity, injection volume) and provided that system connection (tubing, unions) was optimal, the influence of different HPLC-ICP-MS interfaces on extra column dispersion could be investigated. Several pneumatic nebulizers i.e. PFA-ST, MCN6000, AriMist, direct injection high efficiency nebulizer (DIHEN), CEI-100 and a costume made PFA005 nebulizer were tested. It could be shown that the contribution to extra column dispersion is minimal for the DIHEN interface. As a major advantage most of the microbore and capillary hyphenations were not prone to signal suppression due to organic solvents. The 1 mm id microbore set-up employing flow rates of 60 μL min−1 offered the best compromise in terms of sensitivity, peak dispersion, resolution, peak asymmetry and compatibility with organic solvent.

Toxic arsenic compounds in environmental samples: Speciation and validation

Speciation of arsenic (As) compounds in environmental samples requires rigorous analytical procedures at each stage of sample collection, treatment and measurement. In view of the very high number of As species occurring in environmental samples, this article deals with only those that are considered toxic, including As-containing warfare agents and arsenobetaine (AB), because this is the most abundant As species in marine fish. With the exception of the tetramethylarsonium ion, acute toxicity generally decreases with increasing degree of methylation. The warfare agents considered are mostly phenylarsenic compounds. During storage, events, such as changes in oxidation state or induced by microbial activity or losses by volatilization or adsorption, have to be avoided. Extraction of arsenicals from soils and sediments is still a critical step that is not very well controlled. As long as marine animals are considered, extraction efficiencies >90% are commonly obtained [K.A. Francesconi, D. Kuehnelt, Analyst (Cambridge, UK) 129 (2004) 373]. This is logical, since marine animals are rich in AB, and this small molecule is soluble in water and methanol and in mixtures of them. In terrestrial organisms and plants, extraction efficiencies can be much lower and vary according to the extraction conditions. Since most environmental As species are generally present in soluble forms, liquid-separation techniques, first high-performance liquid chromatography (HPLC) but also capillary electrophoresis (CE), are most frequently used for separating As species. Fast separations can be performed with narrow-bore reversed-phase HPLC columns. Pre-column derivatizations with hydride generation [e.g., As(III), As(V), MA, DMA] and mercaptans/dimercaptans (e.g., Clarks I and II and Pfiffikus) allow separation of the arsenicals by gas chromatography (GC). The most popular detectors are inductively coupled mass spectrometry (ICP–MS) and atomic fluorescence spectrometry (AFS), especially after HPLC separation and hydride formation, increasingly replacing atomic absorption spectrometry (AAS), while HPLC–MS or HPLC–MS–MS (HPLC–MS2) is the detector of choice for (less toxic) arsenosugars and arsenolipids. For phenylarsenic warfare species, ultraviolet (UV) detection is also still used frequently. Finally, as other measurement fields, speciation analysis requires the availability of suitable reference materials (RMs) for verification of accuracy and quality assurance. We address these important aspects although the number of certified RMs is rather limited.

Enantiomeric separation of dansyl amino acids using macrocyclic antibiotics as chiral mobile phase additives by narrow-bore high-performance liquid chromatography.

Seven macrocyclic antibiotics were evaluated as chiral selectors for the enantiomeric separation of 11 dansyl amino acids using narrow-bore high-performance liquid chromatography (HPLC). The macrocyclic antibiotics were incorporated as mobile phase additives to determine the enantioselective effects on the chiral analytes. The resolution and capacity factor (k') of each analyte were assessed while varying the structure of macrocyclic antibiotic and the mobile phase buffer pH. The selectivity of the chiral selectors was measured as a function of changes in these parameters. All 11 dansyl amino acids were separated by at least one of the chiral selectors. Three-dimensional computer modeling of the more effective chiral selectors illustrated the importance of macrocyclic antibiotic structure concerning stereospecific analyte interaction.

Development of a liquid chromatographic/tandem mass spectrometric assay for a new endothelin receptor antagonist, and its application to dog plasma samples generated after simultaneous i.v. and p.o. administration of the unlabeled and deuterium-labeled forms of this antagonist.

An assay was developed and applied to determine the bioavailability of a new endothelin receptor antagonist after simultaneous p.o. and i.v. administration of the drug and its stable isotope-labeled analogue. The drug, its main metabolite and the stable isotope-labeled analogues of the drug and the main metabolite were quantified in dog plasma samples using a structural analogue as internal standard. In addition to the calculation of the bioavailability, the formation of the metabolite after p.o. and i.v. administration could be followed independently. The assay covered the concentration range 0.25-1000 ng ml(-1) using sample aliquots of only 50 micro l. Plasma samples were processed after protein precipitation with on-line solid-phase extraction, narrow-bore high-performance liquid chromatography and subsequent tandem mass spectrometric detection. Detection was accomplished with ionspray in the positive ion selected reaction monitoring mode. The inter-assay precision and accuracy of the assay were in the range 4.7-14.2% and 90.3-113.3%, respectively, and the intra-assay precision and accuracy were in the range 1.4-11.5% and 88.4-112.5%, respectively. The fragmentation of the drug was investigated and showed an unexpected shift of a methyl group. Data from MS(n), medium-resolution exact mass tandem mass spectrometry and H-D exchange experiments were employed to clarify the mechanism.

Quantification of metronidazole in small-volume biological samples using narrow-bore high-performance liquid chromatography

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of metronidazole in small volumes of rat plasma, gastric aspirate and gastric tissue. The extraction procedure involves liquid–liquid extraction and a protein precipitation step. A microbore Hypersil ODS 3 μm (150×2.1 mm I.D.) column was used with a mobile phase consisting of acetonitrile–aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90). The column temperature was at 25°C and the detection was by UV absorbance at 317 nm. The limit of detection was 0.015 μg ml −1 for gastric juice aspirate and plasma and 0.010 μg g −1 for gastric tissue (equivalent to 0.75 ng on-column). The method was linear up to a concentration of 200 μg ml −1 for plasma and gastric juice aspirate and up to 40 μg g −1 for tissue, with inter- and intra-day relative standard deviations less than 14%. The measured recovery was at least 78% in all sample matrices. The method proved robust and reliable when applied to the measurement of metronidazole in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.

Identification of anthocyanins in berries by narrow-bore high-performance liquid chromatography with electrospray ionization detection.

Qualitative determination of anthocyanins in extracts of red fruits by narrow-bore HPLC/ESI-MS was carried out. This method was used to investigate anthocyanin contents of black bilberry (Vaccinium myrtillus L.), blackberry (Rubus sp.), and mulberry (Morus nigra). An ultraviolet diode array and a mass spectrometer with ESI source were used for detection. Anthocyanin identifications were made by using retention time data and UV-vis and mass spectra and comparing them with those of commercially available standard compounds. The method allowed the identification of fourteen anthocyanins in black bilberry extract, six anthocyanins in blackberry extract, and five anthocyanins in mulberry extract.

Narrowbore high-performance liquid chromatography for the simultaneous determination of sildenafil and its metabolite UK-103,320 in human plasma using column switching

A fully automated narrowbore high-performance liquid chromatography method with column switching was developed for the simultaneous determination of sildenafil and its active metabolite UK-103,320 in human plasma samples without pre-purification. Diluted plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation occurred to remove proteins and concentrate target substances using 15% acetonitrile in 20 m M phosphate solution (pH 7). The drug molecules eluted from the MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by a valve switching step. The substances enriched in the intermediate column were eluted and separated on a phenyl-hexyl column (100×2 mm I.D.) using 36% acetonitrile in 10 m M phosphate solution (pH 4.5) when the valve status was switched back. The method showed excellent sensitivity (detection limit of 10 ng/ml), good precision (RSD≤2.3%) and accuracy (bias: ±2.0%) and speed (total analysis time 17 min). The response was linear ( r 2≥0.999) over the concentration range 10–1000 ng/ml.

Microdialysis sampling of the isothiazolone, PD-161374, and its thiol and disulfide metabolites

A method based on microdialysis sampling combined with high-performance liquid chromatography (HPLC) has been developed for monitoring the anti-HIV agent PD-161374 (isothiazolone) and its thiol and disulfide metabolites in blood. It was demonstrated that unlike blood withdraw and extraction, microdialysis sampling can preserve the distribution among the isothiazolone and its metabolites in blood. The use of a narrow-bore HPLC system, combined with the relatively high probe extraction efficiency (approximately 50%) from the flexible probe design in this work, allows the direct and quantitative determination of the drug and its major metabolites at submicromolar level.

Narrow-bore high-performance liquid chromatography in combination with ionspray tandem mass spectrometry for the determination of the substance P receptor antagonist ezlopitant and its two active metabolites in plasma

A simple, but highly sensitive and specific, assay was developed for the quantitative determination of ezlopitant and its two active metabolites in human plasma using narrow-bore reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray tandem mass spectrometry (ES–MS–MS). Ezlopitant, its two pharmacologically active metabolites, an alkene analogue (CJ-12 458) and a benzyl alcohol analogue (CJ-12 764), and their corresponding trideuterated internal standards (I.S.), were extracted from plasma with methyl tert.-butyl ether (MTBE). The dried MTBE extracts were reconstituted and analyzed using a narrow-bore (2.1 mm I.D.) YMC basic HPLC column and a mobile phase of acetonitrile–20 m M ammonium acetate, pH 5 (60:40, v/v). Column effluent was monitored by pneumatically assisted electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to product ions was used to quantify ezlopitant and its two active metabolites. The assay exhibited a linear dynamic range of 0.1–100 ng/ml. Average absolute recoveries from plasma were approximately 71, 80 and 99% for ezlopitant and its two active metabolites CJ-12 485 and CJ-12 764, respectively. The precision (RSD %) and accuracy (Deviation %) values for the method were within ±12% and ±15%, respectively, for all analytes. Sample analysis times were less than 5 min from one injection to the next. The assay proved to be suitable for pharmacokinetics studies.

Simultaneous determination of aceclofenac and diclofenac in human plasma by narrowbore HPLC using column-switching

A fully automated narrowbore high performance liquid chromatography (HPLC) with column-switching was developed for the simultaneous determination of aceclofenac and diclofenac from human plasma samples. Plasma sample (100 μl) was directly introduced onto a Capcell Pak MF Ph-1 column (20×4 mm I.D.) where primary separation was occurred to remove proteins and concentrate target substances using acetonitrile–potassium phosphate (pH 7, 0.1 M) (14:86, v/v). The drug molecules eluted from MF Ph-1 column were focused in an intermediate column (35×2 mm I.D.) by the valve switching step. The substances enriched in intermediate column were eluted and separated on the narrowbore phenyl–hexyl column (100×2 mm I.D.) using acetonitrile–potassium phosphate (pH 7, 0.02M) (33:67, v/v) when the valve status was switched back to A position. The method showed excellent sensitivity (detection limit of 10 ng ml −1) with small volume of samples (100 μl), good precision and accuracy, and speed (total analysis time 17 min) without any loss in chromatographic efficiency. The response was linear ( r 2≥0.999) over the concentration range of 50–10000 ng ml −1.

High-speed separation of arsenic compounds using narrow-bore high-performance liquid chromatography on-line with inductively coupled plasma mass spectrometry

The growing importance of arsenic speciation analysis has led to the development of a wide range of high-performance liquid chromatographic based hyphenated techniques. During the present study a method was developed for the high-speed separation of several biologically and environmentally important arsenic compounds. The method is based on the use of an octadecyldimethylsilyl reversed-phase narrow-bore HPLC column. Separation of anionic arsenic species [arsenite (Aite), dimethylarsinic acid (DMAA), monomethylarsonic acid (MMAA), and arsenate (Aate)] can be achieved using a mobile phase containing 5 mM tetrabutylammonium hydroxide as the ion-pairing reagent, at pH 6.0, in less than 2 min, when employing a flow rate of 0.7 ml min−1. Adding 4-hydroxyphenylarsonic acid as the internal standard prolongs the total separation time by 30 s. On-line coupling with inductively coupled plasma mass spectrometry affords high sensitivity, as well as low limits of detection (low ppb or pg of arsenic). The influence of mobile phase pH and ion-pairing reagent concentration on the separation efficiency was studied. A loss of resolution occurs with increasing ion-pairing reagent concentration; the optimum pH is between 6.0 and 6.2. The ion-pair reversed-phase narrow-bore HPLC-ICP-MS method was subsequently applied to the speciation of arsenic in wine and kelp samples. Aite at trace levels was found to be the only arsenic species present in several wines. Average spike recoveries for Aite, Aate, MMAA and DMAA were 95 ± 3, 94 ± 5, 98 ± 1 and 92 ± 1%, respectively, for all wines examined. The method was also used for the speciation of four arsenosugars and DMAA in a kelp powder extract.

Narrowbore high performance liquid chromatography of berberine and palmatine in crude drugs and pharmaceuticals with ion-pair extraction using cobalt thiocyanate reagent

For the simultaneous determination of berberine and palmatine from Phellodendri Cortex, Coptidis Rhizoma and pharmaceuticals, a new narrowbore HPLC method was developed with a simple and selective sample clean-up using cobalt thiocyanate reagent. Samples were sonicated with 2% hydrochloric acid for 30 min and protoberberine-type alkaloids in the resulting mixture were extracted with cobalt thiocyanate reagent and dichloroethane. The aliquot of dichloroethane layer was evaporated and the residue was dissolved for HPLC analysis. The recoveries of berberine and palmatine from Phellodendri Cortex, and Coptidis Rhizoma were better than 90%. Calibration curves for berberine and palmatine were linear over the concentration range of 0.1–50 μg/ml. Limits of quantitation for berberine and palmatine were 0.5 ng. This sample preparation process can be used for the identification of protoberberine-type alkaloids from crude drugs and oriental pharmaceutical preparations.

Highly Sensitive Determination of Histamine by Narrow-Bore High-Performance Liquid Chromatography Using Postcolumn Fluorescence Detection

Highly Sensitive Determination of Histamine by Narrow-Bore High-Performance Liquid Chromatography Using Postcolumn Fluorescence Detection

Determination of a Synthetic Decapeptide in the Plasma of Monkeys and Rats by Narrowbore Highperformance Liquid Chromatography

Analytical methodology utilizing solid-phase extraction and reversed phase narrowbore high-performance liquid chromatography is described for the specific determination of the decapeptide RWJ-47428-099 in the plasma of monkeys and rats. Assay validation included the demonstration of specificity, recoverability of added standard, stability, accuracy, and precision in both matrices. Linearity was demonstrated over the concentration range of 0.031 - 3.0 μg/mL in monkeys and 0.047 - 9.20 μg/mL in rats. This methodology was subsequently implemented in support of preclinical drug metabolism investigations.

Solid Phase Microextraction Coupled to High-Performance Liquid Chromatography

A new interface has been developed to couple the solid phase microextraction (SPME) sampling technique with narrow-bore high-performance liquid chromatography (HPLC). This interface maintains the advantages of SPME as a fast, solvent-free, portable, and inexpensive sampling technique. In addition, it provides possibilities for the analysis of semi- and nonvolatile organic compounds in water, an analysis that is difficult to achieve with gas chromatography. Polycyclic aromatic hydrocarbons (PAHs) were selected as test analytes for the system's evaluation. The interface was shown to provide a successful way of coupling SPME with HPLC for the analysis of PAHs.

Development and validation of a high-sensitivity assay for an antipsychotic agent, CP-88,059, with solid-phase extraction and narrow-bore high-performance liquid chromatography.

An analytical method has been developed and validated for the quantitation of CP-88,059 in human serum. The compound and internal standard were extracted from serum by solid-phase extraction with a weak cation-exchange phase. The analytes were resolved from endogenous interferences using narrow-bore (2.1 mm I.D.) C18 reversed-phase HPLC. Column effluent was monitored by UV absorbance detection at 215 nm. The standard curve range was 1 to 250 ng/ml. The accuracy and precision values for the method were within +/- 10% and +/- 15%, respectively. A four-fold detectability enhancement was achieved using a 2.1 mm I.D. HPLC column relative to the more common 4.6 mm I.D. column. A performance comparison was made between the 2.1 mm I.D. column used for validation and a 4.6 mm I.D. column with the same stationary phase.

Detection and quantitation of gadolinium chelates in human serum and urine by high-performance liquid chromatography and post-column derivatization of gadolinium with Arsenazo III

A narrow-bore high-performance liquid chromatography method was developed for simultaneous separation of gadolinium diethylenetriaminepentaacetic acid (GdDTPA), the monomethylamide (GdDTPA-MMA) and the bis-methylamide (GdDTPA-BMA) in human serum and urine. The Gd complexes were detected at 658 nm after post-column derivatization with Arsenazo III. The serum samples were ultrafiltrated, whereas the urine samples were centrifuged and diluted before analysis. With an injection volume of 10 μl on a 2.1 mm ID reversed-phase column, the limit of detection of GdDTPA-BMA was calculated as 0.3 μM and 1.1 μM in serum and urine, respectively. The method was validated with respect to GdDTPA-BMA with a limit of quantification set to 2 μM and 10 μM in serum and urine, respectively. The best fit of the calibration curve was obtained using non-linear regression according to the equation Y= A+ BX+ CX 2 in the concentration ranges 2–800 μM and 10–2000 μM of GdDTPA-BMA in serum and urine, respectively. The precision of the method was found to range from 1 to 4% RSD. The recoveries of GdDTPA-BMA spiked in serum and urine were higher than 95% with an RSD equal to or less than 4%. The serum samples were stable for at least 5 months when stored at −70 °C, and the urine samples were stable for a least 6 months when stored at −20 °C.