Large area mapping of non-metallic inclusions in stainless steel by an automated system based on laser ablation

Large area compositional mapping (>6 mm²) using a fast and automated system based on laser-induced plasma spectrometry is presented. The second harmonic of a flat top Nd:YAG laser beam was used to generate a microline plasma on the sample surface. The emitted light from the microline plasma was imaged onto the entrance slit of an imaging spectrograph and was detected by an intensified charge-coupled device to generate a spatially and spectrally resolved data set. Individual LIPS images, each measuring roughly 2500×2500 μm with spatial resolution of 50 μm between adjacent craters and 4.8 μm along the microline are presented. These large area maps were acquired in less than 1 min. Steel samples containing MnS and TiN inclusions were chosen as the most adequate for this study. The results are presented for the characterization of inclusionary material in stainless steel products in terms of morphology, distribution and abundance.     

Combining transmission geometry laser ablation and a non‐contact continuous flow surface sampling probe/electrospray emitter for mass spectrometry based chemical imaging

This paper describes the coupling of ambient pressure transmission geometry laser ablation with a liquid‐phase sample collection into a continuous flow surface sampling probe/electrospray emitter for mass spectrometry based chemical imaging. The flow probe/emitter device was placed in close proximity to the surface to collect the sample plume produced by laser ablation. The sample collected was immediately aspirated into the probe and onto the electrospray emitter, ionized and detected with the mass spectrometer. Freehand drawn ink lines and letters and an inked fingerprint on microscope slides were analyzed. The circular laser ablation area was about 210 µm in diameter and under the conditions used in these experiments the spatial resolution, as determined by the size of the surface features distinguished in the chemical images, was about 100 µm. Published in 2011 by John Wiley & Sons, Ltd.     

Characterization of size‐segregated aerosols using ToF‐SIMS imaging and depth profiling

Size‐segregated particles were collected with a ten‐stage micro‐orifice uniform deposit impactor from a busy walkway in a downtown area of Hong Kong. The surface chemical compositions of aerosol samples from each stage were analyzed using time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) operated in the static mode. The ToF‐SIMS spectra of particles from stage 2 (5.6–10 µm), stage 6 (0.56–1 µm), and stage 10 (0.056–0.1 µm) were compared, and the positive ion spectra from stage 2 to stage 10 were analyzed with principal component analysis (PCA). Both spectral analysis and PCA results show that the coarse‐mode particles were associated with inorganic ions, while the fine particles were associated with organic ions. PCA results further show that the particle surface compositions were size dependent. Particles from the same mode exhibited more similar surface features. Particles from stage 2 (5.6–10 µm), stage 6 (0.56–1 µm), and stage 10 (0.056–0.1 µm) were further selected as representatives of the three modes, and the chemical compositions of these modes of particles were examined using ToF‐SIMS imaging and depth profiling. The results reveal a non‐uniform chemical distribution from the outer to the inner layer of the particles. The coarse‐mode particles were shown to contain inorganic salts beneath the organics surface. The accumulation‐mode particles contained sulfate, nitrate, ammonium salts, and silicate in the regions below a thick surface layer of organic species. The nucleation‐mode particles consisted mainly of soot particles with a surface coated with sulfate, hydrocarbons, and, possibly, fullerenic carbon. The study demonstrated the capability of ToF‐SIMS depth profiling and imaging in characterizing both the surface and the region beneath the surface of aerosol particles. It also revealed the complex heterogeneity of chemical composition in size and depth distributions of atmospheric particles. Copyright © 2014 John Wiley & Sons, Ltd.     

Cluster TOF‐SIMS imaging of human skin remains: analysis of a South‐Andean mummy sample

A skin sample from a South‐Andean mummy dating back from the XI^th century was analyzed using time‐of‐flight secondary ion mass spectrometry imaging using cluster primary ion beams (cluster‐TOF‐SIMS). For the first time on a mummy, skin dermis and epidermis could be chemically differentiated using mass spectrometry imaging. Differences in amino‐acid composition between keratin and collagen, the two major proteins of skin tissue, could indeed be exploited. A surprising lipid composition of hypodermis was also revealed and seems to result from fatty acids damage by bacteria. Using cluster‐TOF‐SIMS imaging skills, traces of bio‐mineralization could be identified at the micrometer scale, especially formation of calcium phosphate at the skin surface. Mineral deposits at the surface were characterized using both scanning electron microscopy (SEM) in combination with energy‐dispersive X‐ray spectroscopy and mass spectrometry imaging. The stratigraphy of such a sample was revealed for the first time using this technique. More precise molecular maps were also recorded at higher spatial resolution, below 1 µm. This was achieved using a non‐bunched mode of the primary ion source, while keeping intact the mass resolution thanks to a delayed extraction of the secondary ions. Details from biological structure as can be seen on SEM images are observable on chemical maps at this sub‐micrometer scale. Thus, this work illustrates the interesting possibilities of chemical imaging by cluster‐TOF‐SIMS concerning ancient biological tissues. Copyright © 2012 John Wiley & Sons, Ltd.     

Combined chemical and topographic imaging at atmospheric pressure via microprobe laser desorption/ionization mass spectrometry–atomic force microscopy

The operational characteristics and imaging performance are described for a new instrument comprising an atomic force microscope coupled with a pulsed laser and a linear ion trap mass spectrometer. The operating mode of the atomic force microscope is used to produce topographic surface images having sub‐micrometer spatial and height resolution. Spatially resolved mass spectra of ions, produced from the same surface via microprobe‐mode laser desorption/ionization at atmospheric pressure, are also used to create a 100 × 100 µm chemical image. The effective spatial resolution of the image (～2 µm) was constrained by the limit of detection (estimated to be 10⁹–10¹⁰ molecules) rather than by the diameter of the focused laser spot or the step size of the sample stage. The instrument has the potential to be particularly useful for surface analysis scenarios in which chemical analysis of targeted topographic features is desired; consequently, it should have extensive application in a number of scientific areas. Because the number density of desorbed neutral species in laser desorption/ionization is known to be orders‐of‐magnitude greater than that of ions, it is expected that improvements in imaging performance can be realized by implementation of post‐ionization methods. Published in 2009 by John Wiley & Sons, Ltd.     

Continuous flow infrared matrix‐assisted laser desorption electrospray ionization mass spectrometry

Continuous flow infrared matrix‐assisted laser desorption electrospray ionization (CF IR MALDESI) mass spectrometry was demonstrated for the on‐line analysis of liquid samples. Samples in aqueous solution were flowed through a 50 µm i.d. fused‐silica capillary at a flow rate of 1–6 µL/min. As analyte aqueous solution flowed through the capillary, a liquid sample bead formed at the capillary tip. A pulsed infrared optical parametric oscillator (OPO) laser with wavelength of 2.94 µm and a 20 Hz repetition rate was focused onto the capillary tip for sample desorption and ablation. The plume of ejected sample was entrained in an electrospray to form ions by MALDESI. The resulting ions were sampled into an ion trap mass spectrometer for analysis. Using CF IR MALDESI, several chemical and biochemical reactions were monitored on‐line: the chelation of 1,10‐phenanthroline with iron(II), insulin denaturation with 1,4‐dithiothreitol, and tryptic digestion of cytochrome c. Copyright © 2010 John Wiley & Sons, Ltd.     

Matrix pre‐coated targets for high throughput MALDI imaging of proteins

We have developed matrix pre‐coated targets for imaging proteins in thin tissue sections by matrix‐assisted laser desorption/ionization mass spectrometry. Gold covered microscope slides were coated with sinapinic acid (SA) in batches in advance and were shown to be stable for over 6 months when kept in the dark. The sample preparation protocol using these SA pre‐coated targets involves treatment with diisopropylethylamine (DIEA)‐H₂O vapor, transforming the matrix layer to a viscous ionic liquid. This SA‐DIEA ionic liquid layer extracts proteins and other analytes from tissue sections that are thaw mounted to this target. DIEA is removed by the immersion of the target into diluted acetic acid, allowing SA to co‐crystallize with extracted analytes directly on the target. Ion images (3–70 kDa) of sections of mouse brain and rat kidney at spatial resolution down to 10 µm were obtained. Use of pre‐coated slides greatly reduces sample preparation time for matrix‐assisted laser desorption/ionization imaging while providing high throughput, low cost and high spatial resolution images. Copyright © 2014 John Wiley & Sons, Ltd.     

Refractive index‐modulated grating in two‐mode planar polymeric waveguide produced by two‐photon polymerization

A polymeric waveguide film was manufactured by spinning the materials on quartz substrate. Two‐photon‐initiated photopolymerization was carried out by tight‐focusing femtosecond laser pulses in the two‐mode planar waveguide. A typical index‐modulated grating of 2.5 × 2 mm areas without morphology was fabricated. The results show that peak‐to‐peak modulation depth of the surface profile of grating region was only about 7 nm. The diffraction efficiency (DE) of the grating with a spacing period 2 µm was 0.17% and the corresponding index modulation reached 5.7 × 10^?3. Copyright © 2007 John Wiley & Sons, Ltd.     

Imaging of metals in biological tissue by laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS): state of the art and future developments

Laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS) is well established as a sensitive trace and ultratrace analytical technique with multielement capability for bioimaging of metals and studying metallomics in biological and medical tissue. Metals and metalloproteins play a key role in the metabolism and formation of metal‐containing deposits in the brain but also in the liver. In various diseases, analysis of metals and metalloproteins is essential for understanding the underlying cellular processes. LA–ICP–MS imaging (LA–ICP–MSI) combined with other complementary imaging techniques is a sophisticated tool for investigating the regional and cellular distribution of metals and related metal‐containing biomolecules. On the basis of successful routine techniques for the elemental bioimaging of cryosections by LA–ICP–MSI with a spatial resolution between 200 and ~10 µm, the further development used online laser microdissection ICP–MSI to study the metal distribution in small biological sample sections (at the cellular level from 10 µm to the submicrometer range). The use of mass spectrometric imaging of metals and also nonmetals is demonstrated on a series of biological specimens. This article discusses the state of the art of bioimaging of metals in thin biological tissue sections by LA–ICP–MSI with spatial resolution at the micrometer scale, future developments and prospects for quantitative imaging techniques of metals in the nanometer range. In addition, combining quantitative elemental imaging by LA/laser microdissection–ICP–MSI with biomolecular imaging by matrix‐assisted laser desorption/ionization–MSI will be challenging for future life science research. Copyright © 2013 John Wiley & Sons, Ltd.     

Near‐field laser ablation inductively coupled plasma mass spectrometry: a novel elemental analytical technique at the nanometer scale

An analytical technique utilizing a near‐field effect (to enhance the incident light energy on the thin tip of an Ag needle) in a laser ablation inductively coupled plasma mass spectrometry (NF‐LA‐ICP‐MS) procedure was developed. To produce the thin needles with a tip diameter in the hundreds of nm range a robust needle etching procedure was established. The ‘sample‐to‐tip’ distance was controlled via the measurement of a tunnel current between the needle and sample surface. The NF‐LA‐ICP‐MS technique thus developed was applied for the analysis of copper isotopic standard reference material NIST SRM 976 and tungsten‐molybdenum alloy NIST SRM 480 in the nm resolution range. The observed craters ranged from 200 nm to about 2 µm in diameter and were dependent on the needle used as well as on the ‘sample‐to‐tip’ distance. The mass spectrometric measurements of ⁶³Cu⁺ ion intensity on NIST SRM 976 showed that using near‐field enhancement in laser ablation allowed a roughly 6‐fold increase in the ion intensity of the analyte when the needle was about 100 nm (and below) from the surface, in contrast to when it was far away (e.g. 10 µm) from the sample. The relative standard deviation (RSD) of the ⁶⁵Cu⁺/⁶³Cu⁺ isotopic ratio measurements by NF‐LA‐ICP‐MS was 3.9% (n = 9). The detection efficiencies obtained for the compared LA‐ICP‐MS and NF‐LA‐ICP‐MS methods were found to be 4.6 * 10^?3 counts per second (cps)/ablated atom and 2.7 * 10^?5 cps/ablated atom, respectively. Copyright © 2008 John Wiley & Sons, Ltd.     

A rapid and sensitive LC‐MS/MS method for the determination of linarin in small‐volume rat plasma and tissue samples and its application to pharmacokinetic and tissue distribution study

A rapid and sensitive LC‐MS/MS method was developed for the determination of linarin in small‐volume rat plasma and tissue sample. Sample preparation was employed by the combination of protein precipitation (PPT) and liquid–liquid extraction (LLE) to allow measurement over a 5‐order‐of‐magnitude concentration range. Fast chromatographic separation was achieved on a Hypersil Gold column (100 × 2.1 mm i.d., 5 µm). Mass spectrometric detection was achieved using a triple‐quadrupole mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. Quantification was performed using selected reaction monitoring of precursor‐product ion transitions at m/z 593 → 285 for linarin and m/z 447 → 271 for baicalin (internal standard). The total run time was only 2.8 min per sample. The calibration curves were linear over the concentration range of 0.4–200 µg/mL for PPT and 0.001–1.0 µg/mL for LLE. A lower limit of quantification of 1.0 ng/mL was achieved using only 20 μL of plasma or tissue homogenate. The intra‐ and inter‐day precisions in all samples were ≤14.7%, while the accuracy was within ±5.2% of nominal values. The validated method has been successfully applied to pharmacokinetic and tissue distribution study of linarin. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of mycotoxins in different food commodities by ultra‐high‐pressure liquid chromatography coupled to triple quadrupole mass spectrometry

A rapid multianalyte‐multiclass method with little sample manipulation has been developed for the simultaneous determination of eleven mycotoxins in different food commodities by using ultra‐high‐pressure liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC/MS/MS). Toxins were extracted from the samples with acetonitrile/water (80:20, v/v) 0.1% HCOOH and, after a two‐fold dilution with water, directly injected into the system. Thanks to the fast high‐resolution separation of UHPLC, the eleven mycotoxins were separated by gradient elution in only 4 min. The method has been validated in three food matrices (maize kernels, dry pasta (wheat), and eight‐multicereal babyfood (wheat, maize, rice, oat, barley, rye, sorghum, millet)) at four different concentration levels. Satisfactory recoveries were obtained (70–110%) and precision (expressed as relative standard deviation) was typically below 15% with very few exceptions. Quantification of samples was carried out with matrix‐matched standards calibration. The lowest concentration successfully validated in sample was as low as 0.5 µg/kg for aflatoxins and ochratoxin A in babyfood, and 20 µg/kg for the rest of the selected mycotoxins in all matrices tested. Deoxynivalenol could be only validated at 200 µg/kg, due the poor sensitivity for this mycotoxin analysis. With only two exceptions (HT‐2 and deoxynivalenol), the limits of detection (LODs), estimated for a signal‐to‐noise ratio of 3 from the chromatograms of samples spiked at the lowest level validated, varied between 0.1 and 1 µg/kg in the three food matrices tested. The method was applied to the analysis of different kinds of samples. Positive findings were confirmed by acquiring two transitions (Q quantification, q confirmation) and evaluating the Q/q ratio. Copyright © 2009 John Wiley & Sons, Ltd.     

Image fusion combining SEM and ToF‐SIMS images

Image fusion allows for the combination of an image containing chemical information but low spatial resolution with a high‐spatial resolution image having little to no chemical information. The resulting hybrid image retains all the information from the chemically relevant original image, with improved spatial resolution allowing for visual inspection of the spatial correlations. In this research, images were obtained from two sample test grids: one of a copper electron microscope grid with a letter ‘A’ in the center (referred to below as the ‘A‐grid’), and the other a Tantalum and Silicon test grid from Cameca that had an inscribed letter ‘C’ (referred to below as the ‘Cameca grid’). These were obtained using scanning electron microscopy (SEM) and time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). Image fusion was implemented with the Munechika algorithm. The edge resolution of the resulting hybrid image was calculated compared with the edge resolution obtained for both the individual ToF‐SIMS and SEM images. The challenges of combining complimentary datasets from different instrumental analytical methods are discussed as well as the advantages of having a hybrid image. The distance across the edge for hybrid images of the A‐Grid and the Cameca grid were determined to be 21 µm and 8 µm, respectively. When these values were compared to the original ToF‐SIMS, SEM and optical microscopy measurements, the fused image had a spatial resolution nearly equal to that obtained in the SEM image for both samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Novel floating‐type low‐energy ion gun for high‐resolution depth profiling in ultrahigh vacuum

A floating‐type low‐energy ion gun (FLIG) has been developed for high‐resolution depth profiling in ultrahigh vacuum (UHV). This UHV‐FLIG allows Ar⁺ ions of primary energy down to 50 eV to be provided with high current intensity. The developed UHV‐FLIG was sufficiently compact, being ～30 cm long, to be attached to a commercial surface analytical instrument. The performance of the UHV‐FLIG was measured by attaching it to a scanning Auger electron microprobe (JAMP‐10, Jeol), the base pressure of which in the analysis chamber was ～1 × 10^?7 Pa. The vacuum condition of ～5 × 10^?6 Pa was maintained during operation of the UHV‐FLIG without a differential pumping facility. Current density ranged from 41 to 138 µA cm^?2 for Ar⁺ ions of primary energy 100–500 eV at the working distance of 50 mm. This ensures a sputtering rate of ～10 nm h^?1 with 100 eV Ar⁺ ions for Si, leading to depth profiling of high resolution in practical use. Copyright © 2003 John Wiley & Sons, Ltd.     

Laser ablation isotope ratio mass spectrometry for enhanced sensitivity and spatial resolution in stable isotope analysis

Stable isotope analysis permits the tracking of physical, chemical, and biological reactions and source materials at a wide variety of spatial scales. We present a laser ablation isotope ratio mass spectrometry (LA‐IRMS) method that enables δ¹³C measurement of solid samples at 50 µm spatial resolution. The method does not require sample pre‐treatment to physically separate spatial zones. We use laser ablation of solid samples followed by quantitative combustion of the ablated particulates to convert sample carbon into CO₂. Cryofocusing of the resulting CO₂ coupled with modulation in the carrier flow rate permits coherent peak introduction into an isotope ratio mass spectrometer, with only 65 ng carbon required per measurement. We conclusively demonstrate that the measured CO₂ is produced by combustion of laser‐ablated aerosols from the sample surface. We measured δ¹³C for a series of solid compounds using laser ablation and traditional solid sample analysis techniques. Both techniques produced consistent isotopic results but the laser ablation method required over two orders of magnitude less sample. We demonstrated that LA‐IRMS sensitivity coupled with its 50 µm spatial resolution could be used to measure δ¹³C values along a length of hair, making multiple sample measurements over distances corresponding to a single day's growth. This method will be highly valuable in cases where the δ¹³C analysis of small samples over prescribed spatial distances is required. Suitable applications include forensic analysis of hair samples, investigations of tightly woven microbial systems, and cases of surface analysis where there is a sharp delineation between different components of a sample. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of salbutamol by direct chiral reversed‐phase HPLC using teicoplanin as stationary phase and its application to natural water analysis

A direct chiral LC‐UV method was optimized for the determination of salbutamol (SAL) β₂‐agonist in environmental water. Two commercially available columns were evaluated: teicoplanin Chirobiotic‐T? (150 × 2.1 mm i.d., 5 µm) and vancomycin Chirobiotic‐V? (150 × 2.1 mm i.d., 5 µm). Finally, teicoplanin chiral stationary phase was selected for SAL enantiomer resolution. In order to preserve its integrity and maintain the column performance for longer time, the use of additives such as triethylamine (TEA) in the mobile phase was avoided. Experimental design was applied to simultaneously evaluate the influence of several parameters involved in enantiomer separation and to establish the conditions for acceptable resolution and performance in short analysis time. Optimum mobile phase was methanol–20 mM ammonium acetate buffer at pH 4.5 (98:2, v/v). A solid‐phase extraction procedure for sample pre‐concentration and clean‐up allowed the determination of chiral SAL residues in natural water samples spiked at low concentrations in the range 1.0–20 ng mL^?1. Reproducible recoveries, between 77 and 98%, were obtained and matrix effect was negligible. Injection of sample solutions at low elution strength permitted the SAL enantioresolution in the natural water complex matrix with satisfactory sensitivity and precision. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitation of acotiamide in rat plasma by UHPLC‐Q‐TOF‐MS: method development,validation and application to pharmacokinetics

A novel, sensitive and selective ultra‐high‐performance liquid chromatography–electrospray ionization mass spectrometry method was developed and validated for the quantification of acotiamide (ACT), a first‐in‐class drug used in functional dyspepsia, in rat plasma. A simple protein precipitation method with acetonitrile as precipitating solvent was used to extract ACT from rat plasma. ACT and an internal standard (mirabegron, IS) were separated on an Agilent poroshell EC C₁₈ column (50 × 3.0 mm, 2.7 µm) using methanol–10 mM ammonium acetate binary gradient mobile phase at a flow rate of 0.4 mL/min over 4 min run time. Detection was performed using target ions of [M + H]⁺ at m/z 451.2010 for ACT and m/z 397.1693 for IS in selective ion mode. The method was validated in the calibration range of 1.31–1000 ng/mL. All the validation parameters were well within the limits. The method demonstrated good performances in terms of intra‐ and inter‐day precision (3.27–12.60% CV) and accuracy (87.96–104.94%). Thus the present ultra‐high‐pressure liquid chromatograhy–high‐resolution mass spectrometry method for determination of ACT in rat plasma, is highly sensitive and rapid with a short run‐time of 4 min, can be suitable for high sample throughput and for large batches of biological samples in pharmacokinetic studies. This method can be extended to measure plasma concentrations of ACT in humans to understand drug metabolism, drug interaction and adverse effects. Copyright © 2015 John Wiley & Sons, Ltd.     

Quantitative distribution analysis of alumina inclusion particles in ferritic stainless steels by using laser‐induced breakdown optical emission spectrometry

This paper describes an analytical method for determining the spatial distribution of alumina inclusion particles in several ferritic stainless steels by laser‐induced breakdown optical emission spectrometry with a single‐shot laser scanned on the sample surface. For this purpose, an irradiation system, which comprised a Q‐switched Nd:YAG laser with an average energy of 50 mJ/pulse and a precisely driven X‐Y‐Z sample stage, was prepared. A Czerny–Turner‐mounting spectrograph equipped with an ICCD detector was employed for a time‐resolved measurement of the laser‐induced breakdown optical emission spectrometry signal. The intensity ratio of Al I 396.152 nm to Cr I 396.368 nm was measured each for the single shot, while the irradiation positions were step‐wise moved in the X‐Y direction and then the same sampling area was repeatedly irradiated by subsequent laser shots in the Z direction. The number of alumina particles was mapped from the intensity ratio of Al/Cr each for the irradiation points in both the lateral and in‐depth directions, enabling the distribution of alumina particles to be presented. The resolution of our measuring system was 40 μm in the lateral position and 6–7 μm in the depth direction, which were mainly determined by the crater size of a laser shot. A typical size of the alumina particles (several μm) was smaller than their resolutions; nevertheless, the suggested method would be still more effective to give the distribution of alumina particles, especially the coarse ones, because of its rapid response for the analytical result. Copyright © 2017 John Wiley & Sons, Ltd.     

Rapid and sensitive LC‐MS method for pharmacokinetic study of vinorelbine in rats

A rapid and sensitive LC‐electrospray ionization‐MS method was developed for determining vinorelbine in rat plasma. A 100 µL plasma sample was treated using a protein precipitation procedure and was chromatographed within 4 min using an Inertsil ODS‐3 C₁₈ (2.1 × 50 mm, 5 µm) column. The selected ion monitoring ions [M + H]⁺ were m/z 779 and m/z 811 for vinorelbine and vinblastine (internal standard), respectively. The method validation showed that the calibration curve for vinorelbine was linear over a concentration range of 1–1000 ng/mL with lower limit of quantification at 1 ng/mL. The method has been successfully applied to pharmacokinetics in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of baicalin,oroxylin A‐7‐O‐glucuronide and wogonoside in rat plasma by UPLC‐DAD and its application in pharmacokinetics of pure baicalin,Radix Scutellariae and Yinhuang granule

A novel UPLC‐DAD method was developed and validated for the simultaneous determination of baicalin (baicalein‐7‐glucuronide, BG), oroxylin A‐7‐O‐glucuronide (OAG) and wogonoside (WG) in rat plasma using rutin as the internal standard. Plasma samples were precipitated using acetonitrile containing 0.1% formic acid. Separation was performed on an Agilent Eclipse Plus C₁₈ column (2.1 × 50 mm, 1.8 µm) using gradient acetonitrile and 0.2% formic acid water solution as mobile phase. The flow‐rate was set at 0.4 mL/min and the eluate was detected at 275 nm. The method was linear over the ranges of 0.075–17.50, 0.050–12.60 and 0.056–14.10 µg/mL for BG, OAG and WG, respectively. The intra‐ and inter‐day precisions were respectively <4.8% and 6.4%. All of the limits of detection of three analytes in rat plasma were 0.01 µg/mL, whereas the limits of quantification were, respectively, 0.035, 0.025 and, 0.025 µg/mL. This assay has been successfully applied to pharmacokinetics of BG, OAG and WG in rats after oral administration of Yinhuang granule (YHG) and comparative pharmacokinetics of BG in rats following oral administration of the pure BG, Radix Scutellariae (RS) or YHG. We speculate that some co‐existing ingredients in RS or YHG may increase the absorption and elimination of BG in rat. This work may be helpful for the quality control of Yinhuang granule. Copyright © 2015 John Wiley & Sons, Ltd.