Microm Particle Research Articles

Separation of PCBs by liquid chromatography on reversed phase sub-2-micron particle columns

A new ultra high-performance liquid chromatography method with UV detection was examined for detection and separation of polychlorinated biphenyls. This included optimization of separation conditions for two model mixtures containing seven and fifteen most relevant congeners, comparison of three types of reversed phase sub-2-micron particle sized columns and assessment of system suitability under the optimized conditions. Calibration curves determined in the range from 0.5 to 50.0 microg/mL exhibited correlation coefficients ranging from 0.997 to 0.999. Lower limits of detection ranged from 0.1 to 0.5 ppm. The most efficient Grace C18 column filled with 1.5 microm particles was then tested to separate the complex commercial mixture Delor 103, where the elution order was confirmed by GC-MS. 13 individual congeners were separated and some of the other co-eluting congeners could be resolved using another separation dimension performed with a mass spectrometry detector. The developed method could be directly applied to the separation of less complex mixtures in aqueous sample matrixes, which are used in general for enzyme degradation studies.

Acid leachable trace metals in sediment cores from Sunderban Mangrove Wetland, India: an approach towards regular monitoring

The paper presents the first document to identify the enrichment pattern of acid leachable trace metals (ALTMs) such as Fe, Mn, Cr, Cu, Ni, Pb, Cd, Co, Mo, Ag, As and Ba and their relationship with sediment quality parameters (pH, organic carbon, carbonates and texture) in core sediments (<63 microm particle size) from Indian Sunderban mangrove wetland, formed at the estuarine phase of the river Hugli (Ganges). Textural analysis reveals an overall predominance of mud. The results indicate that the change in pH values causes coagulation and precipitation of ALTMs. Fe and Mn have fairly close distribution patterns of enrichment in surface layers which might be ascribed to early diagnetic processes. The most prominent feature of ALTMs is the enrichment of Fe, Mn, Cr, Cu, Ni, Pb, and Ba in the surface-subsurface layers in the sediment cores, which is mainly attributed to the intense industrial and agricultural activities as well as drainage of untreated domestic sewage to this coastal region. The ALTMs also indicate their association with organic carbon and Fe-Mn oxyhydroxides. The enrichment is well--supported by the correlation, grouping and clustering of ALTMs in statistical analyses. Anthropogenic Factor values indicated ALTMs enrichment for all trace metals due to intense anthropogenic activities. Overall higher values of ALTMs in sediments in comparison to other Indian coastal regions indicate that they are mainly due to the uncontrolled anthropogenic activities in this mangrove estuarine complex. Statistical analyses suggest that five ALTMs (Cu, Pb, As, Mo, Ba) are attached to the organic particles and the clustering of elements separately also indicates that they are from external source. The result of the present study suggests the need for a regular monitoring program which will help to improve the quality of this potential wetland.

Increasing high‐throughput Discovery bioanalysis using automated selected reaction monitoring compound optimization, ultra‐high‐pressure liquid chromatography, and single‐step sample preparation workflows

QuickQuan is an integrated software package for Thermo Scientific triple quadrupole mass spectrometers that allows users to automate routine operations ranging from method development to data processing. QuickQuan automated optimization of compound-selected reaction monitoring (SRM) transitions by evaluating both positive and negative polarities during an infusion. Whichever mode produces the most intense Q1 scan is then carried to product ion spectra. QuickQuan then writes these SRM methods to a shared network database. The total volume of compound needed is 100 microL infused over approximately 1.6 min. The auto-optimization is carried out in 96-well plates and does not require an operator present. The SRM database was shared between two identical TSQ Quantum mass spectrometers. For data acquisition, QuickQuan automatically created a sequence file complete with a data processing method pre-populated with compound IDs and corresponding SRM transitions. To increase throughput we coupled each Finnigan Quantum with ultra-high-pressure liquid chromatography (uHPLC) accomplished using 4x Ultra Flux quaternary pumps that were designed to handle pressures up to 15 000 psi. The total run time for all analyses was 1.2 min using BEH 1.7 microm particle C18 columns. Further time reductions were realized with sample preparation accomplished using Strata Impact protein precipitation plates which provided an in-well protein crash and 0.20 micron filtering in a one-step process. Pharmacokinetic data turnaround time was significantly improved by combining these three techniques of automated method development with the speed efficiency of uHPLC and a single step in well sample preparation.

Development and Validation of a HPLC-MS-MS Method for Quantification of Metronidazole in Human Plasma

The objective of this study was to describe an analytical validation of a simple, rapid, sensitive, and selective liquid chromatography (LC)-tandem mass spectrometry (MS-MS) method for quantification of metronidazole in human plasma samples from bioavailability studies. The plasma samples were pre-treated by liquid-liquid extraction (LLE) with ethyl acetate. Metronidazole and the internal standard (IS) zidovudine were analyzed by combined reversed-phase LC-MS-MS with positive ion electrospray ionization. Multiple reactions monitoring (MRM) of the transitions were used as follows: m/z 171.97 > 127.97 for metronidazole and m/z 268.08 > 126.96 for IS. The main chromatography conditions used were a Varian C(18) Microssorb model (150 mm x 4.6 mm i.d., 5 microm particle size) column, and the mobile phase was composed of acetonitrile and 10 mM ammonium acetate (80:20, v/v) and 0.1% formic acid. Flow rate used was 1.0 mL/min with splitter. The method was linear over a concentration range of 0.05-8.00 microg/mL. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. The developed method was validated and successfully applied to a pilot bioequivalence study of metronidazole in healthy volunteers.

Effect of different surface treatments on In-Ceram Alumina roughness. An AFM study

The aim of this study was to evaluate the influence of different treatments on the surface roughness of In-Ceram Alumina (VITA, Säckingen, Germany). The ceramic blocks were polished (4000 grit) and sandblasted (SB) with airborne particle abrasion Al(2)O(3); 110 microm particle size; 2.8 bar; 20s. The following treatments were performed: (1) no treatment; (2) Rocatec System (RC): after SB treatment, sandblasting with silica powder; 30 microm particle size; 2.8 bar; 20s. (3) Neodymium:yttrium-aluminium-garnet (Nd:YAG) laser (ND) application of graphite powder (stain) on the ceramic surface and Nd:YAG laser irradiation (distance: 1mm, 100mJ, 20 Hz, 2W, and 141.54J/cm(2)). (4) Nd:YAG laser plus Rocatec System: after ND treatments, RC was applied. Digital images (20 microm x 20 microm) from the surfaces were obtained by means of an AFM microscope in taping mode (Nanoscope IIIa, Digital Instruments). Roughness was measured in 10 microm x 10 microm boxes. Roughness (R(a)) data (nm) were analyzed by one-way ANOVA (p<0.05). No differences in ceramic surfaces roughness occurred after any of the tested treatments (p=0.54). High-alumina ceramic surface roughness was not increased with any of the tested treatments.

Computational Simulations of Magnetic Particle Capture in Arterial Flows

The aim of Magnetic Drug Targeting (MDT) is to concentrate drugs, attached to magnetic particles, in a specific part of the human body by applying a magnetic field. Computational simulations are performed of blood flow and magnetic particle motion in a left coronary artery and a carotid artery, using the properties of presently available magnetic carriers and strong superconducting magnets (up to B ≈ 2 T). For simple tube geometries it is deduced theoretically that the particle capture efficiency scales as eta sim sqrt{{Mn}_{rm p}}, with Mn p the characteristic ratio of the particle magnetization force and the drag force. This relation is found to hold quite well for the carotid artery. For the coronary artery, the presence of side branches and domain curvature causes deviations from this scaling rule, viz. η ∼ Mn pβ, with β > 1/2. The simulations demonstrate that approximately a quarter of the inserted 4 μm particles can be captured from the bloodstream of the left coronary artery, when the magnet is placed at a distance of 4.25 cm. When the same magnet is placed at a distance of 1 cm from a carotid artery, almost all of the inserted 4 μm particles are captured. The performed simulations, therefore, reveal significant potential for the application of MDT to the treatment of atherosclerosis.

The effect of air abrasion preparation on the shear bond strength of an orthodontic bracket bonded to enamel

The purpose of this study was to determine the method of preparation of enamel which best retains a bonded orthodontic bracket against a shear force. Two hundred and twelve human lower premolars were randomly divided into four equal groups. Group 1 underwent no air abrasion, group 2 received treatment with 25 microm aluminium oxide particles, group 3 with 50 microm particles, and group 4 with 100 microm particles. All groups were treated with a self-etching primer before bonding of an orthodontic bracket. Each tooth was tested in a JJ Lloyd M30K machine to determine the maximum shear force required to dislodge the bracket from the tooth. A one-way analysis of variance test conducted at a 95 percent confidence level (CL) demonstrated that there was a significant difference (P < 0.01) with respect to the four methods of preparation of the enamel surface. An unpaired t-test was then applied at a 95 percent CL. There was no statistically significant difference between groups 1 and 2. There was, however, a statistically significant difference between groups 1 and 3 (P < 0.01), as well as between groups 1 and 4 (P < 0.01). In addition, there was significant difference found between groups 2 and 3 (P < 0.05), groups 2 and 4 (P < 0.01), and groups 3 and 4 (P < 0.05).

Determination of 8‐epi PGF2α concentrations as a biomarker of oxidative stress using triple‐stage liquid chromatography/tandem mass spectrometry

F2-isoprostanes are a family of prostaglandin F2-like compounds that are formed by free-radical-catalyzed peroxidation of arachidonic acid. Several F2-isoprostanes, but in particular 8-epi PGF(2alpha), are widely used as oxidative stress biomarkers. An analytical method based on liquid chromatography with negative electrospray ionization (ESI) coupled to tandem mass spectrometric detection (LC/MS/MS) was developed for the determination of 8-epi PGF(2alpha) concentrations in human plasma, whole blood, erythrocytes and urine. 8-epi PGF(2alpha)-d4, a stable isotope derivative of 8-epi PGF(2alpha), was used as an internal standard (IS). A 50 microL sample was focused on-column and separated on two 3 microm particle size SUPELCOSIL ABZ+Plus HPLC columns (15 cm x 4.6 mm and 7.5 cm x 4.6 mm) connected in series. An Applied Biosystems 4000 Q TRAP LC/MS/MS system with ESI was operated in multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 353.4 --> 193.1 (8-epi PGF(2alpha)), 357.4 --> 197.1 (8-epi PGF(2alpha)-d4), used for quantification. The assay was fully validated and found to have adequate accuracy, precision, linearity, sensitivity and selectivity. The mass limit of detection (mLOD) was 1 pg of analyte eluting from the column. The assay has been successfully applied to the analysis of human plasma, whole blood, erythrocytes and urine samples.

Application of monolithic columns in pharmaceutical analysis. Determination of indomethacin and its degradation products

Comparison of HPLC methods using conventional particle-based and monolithic columns for determination of indomethacin and its two degradation products, viz. 5-methoxy-2-methylindoleacetic acid and 4-chlorobenzoic acid, was carried out. Ketoprofen was used as an internal standard for data evaluation throughout the study. Conventional separation was based on analytical column Zorbax SB-Phenyl (75x4.6 mm; 3.5 microm particles) used with a mobile phase composition of acetonitrile and phosphoric acid 0.2% (50:50, v/v) and isocratic flow at 0.6 mL/min. Three different lengths of Chromolith columns RP-18e (25x4.6 mm, 50x4.6 mm, and 100x3 mm) were tested with respect to the validation parameters peak asymmetry, resolution, height equivalent to a theoretical plate, repeatability, and after optimisation compared to values obtained using a conventional Zorbax column. The developed methods were used to determine all three compounds in a pharmaceutical formulation--Indobene gel. Chromatographic parameters were comparable to those of a conventional particle-based column. The analysis time was shortened as expected (retention times were lowered by a factor of two). Moreover, the repeatability of peak areas and retention times obtained with a 50 mm monolithic column was greatly improved (RSD values were lower than 0.40%).

Fused‐core, sub‐2 μm packings, and monolithic HPLC columns: a comparative evaluation

Three different HPLC column technologies (i.e., monolith, fused-core particles, and sub-2 microm particles) were evaluated, comparing van Deemter plots, speed of analysis, back pressure, and mobile phase consumption. Very high linear velocities (approximately 12 mm/s) were achieved with the monolithic column using modest pressure (110 bar) at the expense of high mobile phase consumption. The minimum plate height of the monolith was similar to that of a 3 microm-particle packed column (i.e., h = 8 microm), operated at optimal linear velocities; the monolithic column showed substantially lower mass transfer dependence, however. The 2.7 microm fused-core packing material yielded efficiencies closer to the sub-2 microm material than to the 3 microm-particle packed column and could be operated at high flow rates. The fused-core column was able to achieve linear velocities similar to those attained on the sub-2 microm column, staying below 620 bar instead of almost near 1030 bar required by the sub-2 microm material. The lack of pH stability of the monolithic column prevented its use to separate basic compounds (i.e., tricyclic antidepressants) at high pH. Best separation of these components at high pH was achieved using the column packed with 1.7 microm hybrid material.

HPLC of basic drugs using non‐aqueous ionic eluents: evaluation of a 3 μm strong cation‐exchange material

HPLC columns packed with 3 microm particle size HPLC Technology Techsphere SCX (propylsulfonic acid-modified) silica offer considerable advantages over 5 microm SCX packings in the analysis of basic drugs using 100% methanol eluents containing an ionic modifier such as ammonium perchlorate. The basic drugs studied included clozapine and norclozapine, olanzapine, quinine and quinidine, and amitriptyline, nortriptyline, imipramine and desipramine. The 3 microm column was not only more efficient for a given column length compared with 5 microm materials, but also elution times were less, a phenomenon observed in reversed-phase systems. The high efficiencies and excellent peak shapes obtained with the 3 microm SCX-modified packing together with the relatively low back-pressures attained show that such materials deserve serious consideration by laboratories involved in the analysis of basic drugs. Manufacturers should offer such packings as a matter of routine. Alternative ionic modifiers such as ammonium acetate are available for use with mass spectrometric detection if required.

Stability-Indicating Reversed-Phase Liquid Chromatographic Methods for the Determination of Aconitine and Piperine in a Polyherbal Formulation

Simple and rapid stability-indicating HPLC methods were developed for the individual analysis of aconitine (ACN) and piperine (PIN) in Mahamrutynjaya rasa, an herbal dosage form containing Aconitum ferox, Piper nigrum, and Piper longum in combination. Separation of the ACN from its major and minor degradation products was successfully achieved on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size), with isocratic elution using a mixture of acetonitrile-KH2PO4 buffer (10 mM, pH 8 +/- 0.1; 50 + 50, v/v) at flow rate of 0.7 mL/min with UV detection at 227 nm. PIN separation was performed on a reversed-phase C18 column (250 x 4.6 mm id, 5 microm particle size), with isocratic elution in acetonitrile-KH2PO4 buffer (10 mM, pH 7 +/- 0.1; 35 + 65, v/v) at a flow rate of 1 mL/min with UV detection at 343 nm. The methods were validated with respect to linearity, precision, accuracy, specificity, system suitability, and robustness. The responses were linear in the drug concentration range of 10-100 microg/mL for both ACN and PIN. The percent recoveries of both the markers from a mixture of degradation products were in the range between 98-101%. The utility of the procedures was verified by their application to marketed formulations that were subjected to accelerated degradation studies. The methods could distinctly separate the drug and degradation products. The products formed in the marketed tablets were similar to those formed in the laboratory during stress studies.

Fast liquid chromatography separation and multiple‐reaction monitoring mass spectrometric detection of neurotransmitters

We describe here the fast LC-MS/MS separation of a mixture of neurotransmitters consisting of dopamine, epinephrine, norepinephrine, 3,4-dihydroxybenzylamine (DHBA), salsolinol, serotonin, and gamma-aminobutyric acid (GABA). The new UltiMate 3000 Rapid Separation system (RSLC) was successfully coupled to the 4000 QTRAP mass spectrometer operating in multiple-reaction monitoring (MRM) mode. The separation was attained using a 100 mm length, 2.2 microm particle size Acclaim column at a flow rate of 0.5 mL/min. The column back pressure was 350 bar, while the total run time including column re-equilibration was 5.2 min. The peak resolution was minimally affected by the fast separation. The RSLC-MRM separation was found to have a precision range based on peak area for 50 replicate runs of 2-5% CV for all analytes, and the reproducibility of the retention time for all analytes was found to range from 0-2% CV. The described method represents an almost seven times shorter analysis time of neurotransmitters using LC/MRM which is very useful in screening large quantities of biological samples for various neurotransmitters.

Immunohistochemical Characterization of Specific Inflammatory Tissue Reactions following Embolization with Four Different Spherical Agents in the Minipig Kidney Model

To evaluate the immunohistochemical inflammatory reaction after porcine renal embolization with the new spherical embolic agent Embozene and to compare it with other spherical embolic agents. After superselective porcine renal embolization (40 pigs) with different sizes of embolic agents (Embozene, Embosphere, Bead Block, Contour SE), tissue arrays were obtained (size ranges, 40-120 microm, 100-300 microm, 500-700 microm, 700-900 microm). After immunostaining for CD subtyping (CD45 and CD68) and cytokines (C-reactive protein [CRP] and interleukin-1 beta), a semiquantitative immunoreactivity score was calculated for each marker: intensity of staining was scored between 0 (negative) and 3 (intensive) and extent of staining between 0 and 4 (>80%), indicating the percentage of positive staining. The intensity score (0-3) was multiplied by the extent of staining score (0-4), resulting in a semiquantitative immunoreactivity score (0-12). Analysis of cellular expression profiles (ie, CD45, CD68) revealed a significantly higher inflammatory score 4 weeks after embolization with Embosphere 100-300 microm particles than after embolization with Embozene, Bead Block, and Contour SE. After 12 weeks, the Embosphere 100-300 microm score decreased. Analysis of CRP expression showed similar results, with a significantly higher score 4 weeks after embolization with Embosphere 100-300 microm. In the size class used most frequently for uterine artery emboliation (500-700 microm), all scores were low (<2.5) and there was no significant difference among particle types. Pronounced immunomarker expression was seen 4 weeks after embolization with small Embosphere particles. However, in general, modern spherical embolic agents cause a fairly low level of inflammatory reaction. In the present experimental setting, which is highly sensitive for specific tissue-to-agent reactivity, Embozene presented with low inflammatory results.

Fast chiral separation of drugs using columns packed with sub‐2 μm particles and ultra‐high pressure

The use of columns packed with sub-2 microm particles in liquid chromatography with very high pressure conditions (known as UHPLC) was investigated for the fast enantioseparation of drugs. Two different procedures were evaluated and compared using amphetamine derivatives and beta-blockers as model compounds. In one case, cyclodextrins (CD) were directly added to the mobile phase and chiral separations were carried out in less than 5 min. However, this strategy suffered from several drawbacks linked to column lifetime and low chromatographic efficiencies. In the other case, the analysis of enantiomers was carried out after a derivatization procedure using two different reagents, 2,3,4-tri-O-acetyl-alpha-D-arabinopyranosyl isothiocyanate (AITC) and N-alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent). Separation of several amphetamine derivatives contained within the same sample was achieved in 2-5 min with high efficiency and selectivity. The proposed approach was also successfully applied to the enantiomeric purity determination of (+)-(S)-amphetamine and (+)-(S)-methamphetamine. Similar results were obtained with beta-blockers, and the separation of 10 enantiomers was carried out in less than 3 min, whereas the individual separation of several beta-blocker enantiomers was performed in 1 min or less.

Fast log P determination by ultra-high-pressure liquid chromatography coupled with UV and mass spectrometry detections

Ultra-high-pressure liquid chromatography (UHPLC) systems able to work with columns packed with sub-2 microm particles offer very fast methods to determine the lipophilicity of new chemical entities. The careful development of the most suitable experimental conditions presented here will help medicinal chemists for high-throughput screening (HTS) log P(oct) measurements. The approach was optimized using a well-balanced set of 38 model compounds and a series of 28 basic compounds such as beta-blockers, local anesthetics, piperazines, clonidine, and derivatives. Different organic modifiers and hybrid stationary phases packed with 1.7-microm particles were evaluated in isocratic as well as gradient modes, and the advantages and limitations of tested conditions pointed out. The UHPLC approach offered a significant enhancement over the classical HPLC methods, by a factor 50 in the lipophilicity determination throughput. The hyphenation of UHPLC with MS detection allowed a further increase in the throughput. Data and results reported herein prove that the UHPLC-MS method can represent a progress in the HTS-measurement of lipophilicity due to its speed (at least a factor of 500 with respect to HPLC approaches) and to an extended field of application.

Separation of natural product using columns packed with Fused‐Core particles

Three HPLC columns packed with 3 microm, sub-2 microm, and 2.7 microm Fused-Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express C18 column packed with Fused-Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3 C18 column packed with 3 microm particles. Column lot-to-lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity BEH column packed with sub-2 microm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high-efficiency and high-speed separation to be performed using conventional HPLC instrumentation.

Development and Validation of a Simple Method for Routine Analysis of Ractopamine Hydrochloride in Raw Material and Feed Additives by HPLC

A simple method was optimized and validated for determination of ractopamine hydrochloride (RAC) in raw material and feed additives by HPLC for use in quality control in veterinary industries. The best-optimized conditions were a C8 column (250 x 4.6 mm id, 5.0 microm particle size) at room temperature with acetonitrile-100 mM sodium acetate buffer (pH 5.0; 75 + 25, v/v) mobile phase at a flow rate of 1.0 mL/min and UV detection at 275 nm. With these conditions, the retention time of RAC was around 5.2 min, and standard curves were linear in the concentration range of 160-240 microg/mL (correlation coefficient > or = 0.999). Validation parameters, such as selectivity, linearity, limit of detection (ranged from 1.60 to 2.05 microg/mL), limit of quantification (ranged from 4.26 to 6.84 microg/mL), precision (relative standard deviation < or = 1.87%), accuracy (ranged from 96.97 to 100.54%), and robustness, gave results within acceptable ranges. Therefore, the developed method can be successfully applied for the routine quality control analysis of raw material and feed additives.

High‐performance liquid chromatography and LC‐ESI‐MS method for identification and quantification of two isomeric polyisoprenylated benzophenones isoxanthochymol and camboginol in different extracts of Garcinia species

A rapid, sensitive and simple reverse-phase high-performance liquid chromatography-electrospray ionization mass spectrometric method has been developed for the identification and quantification of two isomeric polyisoprenylated benzophenones, isoxanthochymol and camboginol, in the extracts of the stem bark, seeds and seed pericarps of Garcinia indica and in the fruit rinds of Garcinia cambogia. The separation of isoxanthochymol and camboginol was achieved on a Perkin Elmer RP(8) column (10 x 2.1 mm with 5.0 microm particle size) using a solvent system consisting of a mixture of acetonitrile-water (80:20, v/v) and methanol-acetic acid (99.0:1.0, v/v) as a mobile phase in a gradient elution mode. The limits of detection and quantification were 5 and 10 microg/mL for isoxanthochymol and 50 and 100 microg/mL for camboginol, respectively. The intra- and inter-day precisions were 2.34 and 3.41% for isoxanthochymol and 3.35 and 3.66% for camboginol. The identity of the two isomeric compounds in the samples was determined on a triple quadrupole mass spectrometer with ESI interface operating in the negative ion mode. The method was used to identify and quantify isoxanthochymol and camboginol in the different extracts of two Garcinia species, Garcinia indica and Garcinia cambogia.

Simultaneous determination of six herbal components in intestinal perfusate by high‐performance liquid chromatography

An effective, accurate and reliable HPLC with UV detection method was developed and validated for quantitation of six components: baicalin, berberine hydrochloride, quercetin, kaempferol, isorhamnetin and baicalein in intestinal perfusate using rotundin as an internal standard. The chromatographic separation was performed on a Welchrom-C(18) column (250 x 4.6 mm i.d. with 5.0 microm particle size) with a mobile phase consisting of acetonitrile, water, phosphoric acid and triethylamine (30:70:0.2:0.1,v/v) at a flow rate of 1.0 mL/min and a UV detection at 270 nm. The method had a chromatographic run time of 30 min and excellent linear behavior over the investigated concentration ranges observed with the values of r higher than 0.99 for all the analytes. The lower limit of quantification of the analytical method was 0.09 microg/mL for berberine hydrochloride, quercetin, kaempferol and baicalein and 0.18 microg/mL for baicalin and isorhamnetin. The intra- and inter-day precisions measured at three concentration levels were all less than 10% for all analytes. The bias ranged from -6.91 to 4.33%. The validated method has been successfully applied to investigate the rat intestine absorption profiles of baicalin, berberine hydrochloride, quercetin, kaempferol, isorhamnetin and baicalein.