Quantification of 3‐n‐butylphthalide in beagle plasma samples by supercritical fluid chromatography with triple quadruple mass spectrometry and its application to an oral bioavailability study

A high‐throughput, rapid, sensitive, environmentally friendly, and economical supercritical fluid chromatography with triple quadruple mass spectrometry method was established and validated for the first time to determine a cerebral stroke treatment drug named 3‐n‐butylphthalide in dog plasma. Plasma samples were prepared by protein precipitation with methanol and the analytes were eluted on an ACQUITY UPC^2TM HSS‐C₁₈ SB column (3 × 100 mm, 1.8 μm) maintained at 50°C. The mobile phase comprised supercritical carbon dioxide/methanol (90:10, v/v) at a flow rate of 1.5 mL/min, the compensation solvent was methanol at a flow rate of 0.2 mL/min and the total run time was 1.5 min per sample. The detection was carried out on a tandem mass spectrometer with an electrospray ionization source. Calibration curves were linear over the concentration range of 1.02–1021.00 ng/mL (r² ≥ 0.993) with the lower limit of quantification of 1.02 ng/mL. The intra‐ and inter‐day precision values were below 15% and the accuracy was from 97.90 to 103.70% at all quality control levels. The method was suitable for a pharmacokinetic study of 3‐n‐butylphthalide in beagle dogs.     

Rapid Analysis of 27 Components of Isodon serra by LC–ESI-MS–MS

A new method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry has been developed for simultaneous analysis of 27 components (eighteen diterpenoids, six phenolic acids, and three flavonoids) of Isodon serra, a famous traditional Chinese medicine. Separation on a C₁₈ column was achieved by gradient elution with water and methanol both containing 0.1% formic acid. Identification and quantification of the analytes were achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring (MRM) was used for quantification, with switching of electrospray ion source polarity between positive and negative modes in the same chromatographic run. An information-dependent acquisition (IDA) method was used to trigger product ion scans above the MRM signal threshold so that the 27 compounds could be identified by use of enhanced product-ion scans (EPI). The method was fully validated (for linearity, precision, accuracy, and limits of detection and quantification). The results indicated that this simple method was rapid, specific, and reliable. The method was successfully applied to analysis of 45 batches of Isodon serra samples from different sources, and quantification of the compounds in Isodon serra was achieved.     

A sensitive,high‐throughput,and eco‐friendly analysis of daidzein and its valine carbamate prodrug in rat plasma by supercritical fluid chromatography with tandem mass spectrometry

A valine carbamate prodrug of daidzein was synthesized to improve its bioavailability because of the poor solubility and low permeability of daidzein. To evaluate the pharmacokinetic behavior of the prodrug, a sensitive and high‐throughput method was developed and validated for the simultaneous determination of daidzein and its prodrug in rat plasma. The samples were extracted by ethyl acetate and then analyzed by a supercritical fluid chromatography with electrospray ionization tandem mass spectrometry method. The separation was achieved by an ACQUITY UPC²™ BEH 2‐EP column maintained at 40°C using carbon dioxide (≥99.99%) and methanol within 3.0 min by gradient elution. The mass transition ion pairs were m/z 254.8→136.7, 398.0→254.9, and 271.0→91.07 for daidzein, the prodrug, and genistein, respectively. The calibration curves were linear over the concentration ranges of 2–500 (r > 0.997) and 10.0–5000.0 ng/mL (r > 0.996) with lower limits of quantification of 2 and 10 ng/mL for daidzein and the prodrug, respectively. The intra‐ and interday accuracy and precision were within ±15% for all quality control samples. This developed method enabled high specificity, low cost, low solvent consumption, and a brief analysis time and was successfully applied to a bioavailability evaluation of daidzein and its carbamate prodrug.     

Developing the liquid chromatography-mass spectrometry method for simultaneously quantifying five components in rat serums after oral administration of hawthorn aqueous extracts and its application to a pharmacokinetic study

Hawthorn, one of the widely-used traditional Chinese medicines, has been used to treat dyspepsia, hyperlipidemia, and cardiovascular disease in the clinic. Our previous study revealed that gallic acid, neochlorogenic acid, cryptochlorogenic acid, vitexin, and quercetin were active components of hawthorn. In this study, a simple, precise, and reliable liquid chromatography-mass spectrometry method was developed for the simultaneous quantification of five components in rat serums. The separation was achieved on the Hypersil GOLD C₁₈ column, and the mobile phases consisted of 0.1% acetic acid water and methanol at a flow rate of 0.3 mL/min. The mass spectrometry data acquisition was performed on Q-Extractive-Orbitrap mass spectrometry with an electrospray ionization source in negative ion mode. The proposed liquid chromatography-mass spectrometry method was validated in terms of linearity, intra- and inter-precision, accuracy, recoveries, matrix effects, and stability. Then this newly proposed liquid chromatography-mass spectrometry method was successfully applied to a pharmacokinetic study on rats after oral administration of hawthorn aqueous extracts. This study provided relevant information on the pharmacokinetics of active components of hawthorn and explained the underlying mechanism of their bioactivity.     

Analysis of thiabendazole and procymidone in fruits and vegetables by capillary electrophoresis-electrospray mass spectrometry

A capillary electrophoresis-mass spectrometry method for determining procymidone and thiabendazole in apples, grapes, oranges, pears, strawberries and tomatoes is described. Separation is achieved using a buffer of formic acid-ammonium formate at pH 3.5 with 2% of methanol. Fungicide residues present in the sample are preconcentrated by both solid-phase extraction and injection of large sample volumes into the capillary by a stacking technique, to obtain lower detection limits. Ionization is performed at atmospheric pressure in an electrospray type source and detection is carried out using positive ionization and selected ion monitoring modes. The quantitation limits are 0.005 and 0.05 mg kg(-1), and the mean recoveries are 64 and 75% for thiabendazole and procymidone, respectively, with relative standard deviations below 12% (n=5). Real fruit and vegetable samples are analyzed by the proposed method showing that residues of both fungicides are frequently present.     

Characterization and determination of chlorophacinone in plasma by ion chromatography coupled with ion trap electrospray ionization mass spectrometry

Plasmatic chlorophacinone is commonly measured with liquid chromatographic assay, which convenient but lacks sensitivity and selectivity and usually requires ion pair reagents to reduce the chromatographic tailed peak. In this paper, a novel method using eluent generator reagent‐free ion chromatography coupled with electrospray ionization ion trap mass spectrometric detection for the determination of chlorophacinone in plasma has been developed. After samples were extracted with 10% (v/v) methanol in acetonitrile and cleaned by solid‐phase extraction, chromatographic separation was performed on an IonPac^® AS11 analytical column (250 × 4.0 mm) using 40.0 mmol/L KOH containing 10% (v/v) methanol as organic modifier. Quantification was performed by negative electrospray ionization in multiple reaction monitoring mode. The transition m/z 373 → 201 was for the quantification ion; the transitions m/z 373 → 172 and m/z 373 → 145, as well as the isotope ions m/z 375 and m/z 203, were for the qualitative ions. All the method parameters were validated. It was confirmed that this method can be used in clinical diagnosis and forensic toxicology. Copyright © 2008 John Wiley & Sons, Ltd.     

Characterization of triglycerides in vegetable oils by silver-ion packed-column supercritical fluid chromatography coupled to mass spectroscopy with atmospheric pressure chemical ionization and coordination ion spray

Characterization of triglycerides in vegetable oils was achieved by silver-ion packed-column supercritical fluid chromatography (SI-pSFC) with mass spectrometric detection. Hyphenation was made using commercially available liquid chromatography-mass spectrometry (LC-MS) interfaces without any modification. A make-up fluid was delivered through a T-piece placed before or after the SFC restrictor by means of a high pressure pump. Atmospheric pressure chemical ionization (APCI) and coordination ion spray (CIS) with silver ions were used as ionization modes. Compared to UV detection, the sensitivity was increased by a factor of 100. Both ionization modes are generating similar structural information. Molecular ions [M-H]+ or [M-Ag(-)] are observed in the mass spectra with exception of the saturated triglycerides for which only CIS gives intense molecular ions. The position at which the fatty acids are esterified to the glycerol backbone can be elucidated by pSFC-APCI although it remains speculative whether this is valid for highly unsaturated triglycerides because reference compounds are not available to proof this.     

Fast quantification of flavonoids in <Emphasis Type="Italic">Filipendulae ulmariae flos</Emphasis> by HPLC/ESI-MS using a nonporous stationary phase

A sensitive and rapid method for the quantification of flavonoids in Filipendulae ulmariae flos (Filipendula ulmaria L.) was developed using HPLC with diode array detection coupled to electrospray ionization mass spectrometry (ESI-MS). Separation was achieved on a 1.5-μm nonporous C-18 phase by gradient elution with acetonitrile and 0.03 M acetic acid. Quantification was performed by internal standardization with acacetin. To enhance selectivity, ESI-MS detection was optimized in the negative mode and selected ion monitoring (SIM mode). The text was submitted by the authors in English.     

Determination of selected fatty acids in dried sweat spot using gas chromatography with flame ionization detection

A method is described for the determination of fatty acids in dried sweat spot and plasma samples using gas chromatography with flame ionization detection. Plasma and dried sweat spot samples were obtained from a group of blood donors. The sweat was collected from each volunteer during exercise. Sweat was spotted onto collection paper containing butylated hydroxytoluene. Fatty acids were derivatized with acetyl chloride in methanol to form methyl esters of fatty acids. The fatty acids in dried sweat spot samples treated with butylated hydroxytoluene and stored at –20°C were stable for 3 months. Our results indicate that sweat contains, among fatty acids with short chain, also fatty acids with long chain and unsaturated fatty acids. Linear relationships between percentage content of selected fatty acids in dried sweat spot and plasma were observed.     

Ring-methylation of pyrrole and indole using supercritical methanol

The ring-methylation of pyrrole or indole using supercritical methanol proceeded at 623 K without the further addition of catalysts. Pyrrole produced a mixture of unreacted pyrrole and mono-, di-, tri-, and tetra-methylpyrroles at the reaction time of 8 h. On the other hand, indole was selectively methylated at the C3 position to afford 3-methylindole in 79% yield at the reaction time of 5 h. The ring-methylation of indole using supercritical methanol was claimed to proceed via (1H-indol-3-yl)methanol. The conversion of indole to (1H-indol-3-yl)methanol would be achieved by the electrophilic aromatic substitution between the indol-1-ide (indole anion) and H₂C⁺–OH. The (1H-indol-3-yl)methanol must be reduced to 3-methylindole in the presence of supercritical methanol.     

Supercritical fluid chromatography with post-column addition of supporting electrolyte solution for electrochemical determination of tocopherol and tocotrienol isomers

A supercritical fluid chromatography with electrochemical detection system was developed for the simultaneous determination of tocopherol and tocotrienol isomers. The supercritical fluid chromatography with electrochemical detection system was connected with an additional pump to create a flow path to add a supporting electrolyte solution. The supporting electrolyte solution was mixed with a mobile phase in a post-column fashion, enabling the independent control of the separation and detection. After optimization of the measurement conditions, vitamin E isomers and an internal standard substance (2,2,5,7,8-pentamethyl-6-hydroxychroman) were separated within 30 min using a mixture of supercritical carbon dioxide and methanol (99:1, v/v) as a mobile phase and a cyanopropyl column (4.6 mm inner diameter × 250 mm length, 5 μm). For the electrochemical detection, methanol containing 1.0 mol/L ammonium acetate was used as a supporting electrolyte solution, and the applied potential was set at +0.8 V. This analytical method showed good linearity (5–100 μg/mL) and repeatability (less than 2.5% relative standard deviation, n = 6) and was applicable to the determination of tocopherol and tocotrienol isomers in nutrition supplements.     

A simple and rapid determination of valproic acid in human plasma using a non-porous silica column and liquid chromatography with tandem mass spectrometric detection

A liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay was developed and validated to determine valproic acid in human plasma. The method involved a solid-phase extraction of valproic acid and betamethasone valerate, an internal standard, from plasma and detection using an LC-MS/MS system with electrospray ionization source in negative ion mode. Separation was achieved within 3 min on a non-porous silica column with mobile phase containing ammonium acetate and methanol. Multiple reaction monitoring was utilized for detection monitoring at 142.89-142.89 for valproic acid and 457.21-457.21 for the internal standard. The calibration curve for valproic acid was linear over the range of 0.5-150 microg/mL. The limit of detection was 0.17 microg/mL and the lower limit of quantification was 0.5 microg/mL, when 0.2 mL plasma was used for extraction. The percentage coefficient of validation for accuracy and precision (inter- and intra-day) for this method was less than 9.5% with recovery ranging from 82.3 to 86.9% for valproic acid.     

Direct analysis of free amino acids by mixed‐mode chromatography with tandem mass spectrometry

We developed a straightforward, robust, and relatively fast method for the analysis of amino acids by mixed‐mode high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The method does not involve derivatization and allows the detection of 21 amino acids, representing a wide range of isoelectric points, in less than 40 min. Chromatographic separation was governed by a silica‐based mixed‐mode column providing simultaneous hydrophobic and ion exchange separation mechanisms. The use of tandem mass spectrometry increased selectivity, reducing potential problems associated with poor selectivity in the chromatographic system. For an injection volume of 1 μL, we obtained detection limits <3 μM for the majority of analytes. For all analytes, a linearity of r > 0.99 was obtained, recovery in matrix was >86%, and the retention times were highly reproducible. The method was successfully applied to soil solution and fungal culture samples, demonstrating the advantages in successfully avoiding issues associated with high amounts of substances that may interfere with derivatization‐based methods. This method represents an alternative to derivatization‐based methods and can be applied in areas where sample matrices are highly complex.     

Characterization of constituents in Stellera chamaejasme L. by rapid-resolution liquid chromatography-diode array detection and electrospray ionization time-of-flight mass spectrometry

A reliable and rapid method based on rapid-resolution liquid chromatography-diode array detection (RRLC-DAD) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF/MS) has been developed for the isolation and characterization of multiple constituents in the root of Stellera chamaejasme L., which was extracted by sonication with methanol in an optimized procedure. Separation of the multiple constituents was achieved on an Agilent Zorbax XDB-C18 (50x3.0 mm i.d.; 1.8 microm) column using a gradient elution at a flow rate of 0.4 mL/min. The detection wavelength was 210 nm. Mass spectra were acquired in both positive and negative modes. A formula database of the known chemical constituents in the root of Stellera chamaejasme L. was established by an Agilent software. Twenty-two obvious peaks appeared in the total ion chromatogram and nine of them were characterized by TOF/MS. The RRLC-DAD and ESI-TOF/MS method with ultrasonic extraction would be useful for rapid and effective characterization of chemical constituents in the root of Stellera chamaejasme L.     

Technology for the identification of unusual cis, cis octadecadienoic fatty acids.

In this article methods are presented for the separation and identification of unusual cis, cis dienoic and polygnoic long chain fatty acids. Special emphasis has been laid on the identification of cis, cis octadecadienoic acids. The steps followed are: after transesterification the fatty acid methyl esters are separated by preparative gas chromatography according to chain length followed by argentation chromatography on thin-layer plates. After hydroxylation of the double bonds with osmium tetroxide the polyhydroxy compounds are derivatized to the per-O-trimethylsilyl-ethers. Separation and identification of individual compounds are achieved by combined gas chromatography-mass spectrometry using SCOT columns and low ionization energy.     

Simultaneous analysis of 11 main active components in Cirsium setosum based on HPLC‐ESI‐MS/MS and combined with statistical methods

A novel method based on high‐performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry was developed for simultaneous determination of the 11 major active components including ten flavonoids and one phenolic acid in Cirsium setosum. Separation was performed on a reversed‐phase C₁₈ column with gradient elution of methanol and 0.1‰ acetic acid (v/v). The identification and quantification of the analytes were achieved on a hybrid quadrupole linear ion trap mass spectrometer. Multiple‐reaction monitoring scanning was employed for quantification with switching electrospray ion source polarity between positive and negative modes in a single run. Full validation of the assay was carried out including linearity, precision, accuracy, stability, limits of detection and quantification. The results demonstrated that the method developed was reliable, rapid, and specific. The 25 batches of C. setosum samples from different sources were first determined using the developed method and the total contents of 11 analytes ranged from 1717.460 to 23028.258 μg/g. Among them, the content of linarin was highest, and its mean value was 7340.967 μg/g. Principal component analysis and hierarchical clustering analysis were performed to differentiate and classify the samples, which is helpful for comprehensive evaluation of the quality of C. setosum.     

A sensitive,high‐throughput,and ecofriendly method for the determination of lumefantrine,artemether, and its active metabolite dihydroartemisinin by supercritical fluid chromatography and tandem mass spectrometry

A quick and sensitive supercritical fluid chromatography with tandem mass spectrometry method for the simultaneous determination of lumefantrine, artemether, and its active metabolite dihydroartemisinin in rat plasma was developed and validated. The chromatographic separation was performed on an ACQUITY UPC²™ BEH 2‐EP column within 2.5 min by gradient elution using compressed CO₂ and methanol containing 2 mM ammonium acetate as the mobile phases. Detection was achieved by multiple reaction monitoring using electrospray ionization in the positive ionization mode. For sample preparation, 50 μL of the sample was processed by modified high‐throughput, one‐step protein precipitation using hydrogen peroxide as a stabilizer to protect the endoperoxide‐containing artemisinin derivatives from degradation. The calibration curves were linear over the concentration range of 2.0–1000 ng/mL for both artemether and dihydroartemisinin, and 1.0–5000 ng/mL for lumefantrine. The values of selectivity, lower limit of quantification, linearity, accuracy, precision, matrix effects, stability, and recovery met the acceptable range according to the Food and Drug Administration guidelines. The developed method enables high resolution and speed as well as low cost, low solvent consumption, and short time and was successfully applied to pharmacokinetic studies through the intravenous administration of an artemether–lumefantrine lipid emulsion in rats.     

Gas chromatography of bile acids as their hexafluoroisopropyl ester-trifluoroacetyl derivatives.

Bile acids, such as cholic, chenodeoxycholic, deoxycholic, lithocholic and ursodeoxycholic acids, were allowed to react with hexafluoroisopropanol and tri-fluoracetic anhydride at 37 for 30 min. The resulting derivatives were gas chromatographed on QF-1, with flame ionization detection, and were identified by gas chromatography-mass spectrometry. Separation was good. By using this method, these acids were detected in samples of human duodenal fluid; the ratios of each were 24.4, 41.5, 24.9, 2.3 and 6.9%, respectively.     

Determination of tributyltin and 4-hydroxybutyldibutyltin chlorides in seawater by liquid chromatography with atmospheric pressure chemical ionization-mass spectrometry

A liquid chromatographic method is described for the simultaneous determination of tributyltin (TBT) and the hydroxylated intermediate 4-hydroxybutyldibutyltin (OHBuDBT). Separation was achieved in reverse phase mode on a cyanopropyl-bonded silica column under a gradient elution. Various organic solvents and additives were tested and the optimum composition of the mobile phase contained methanol, water, formic acid and tropolone as a complexing agent. Butyltin compounds were detected with an ion trap mass spectrometer interfaced to a liquid chromatograph with an atmospheric pressure chemical ionization source (LC-APCI-MS). Identification and fragmentation pattern of OHBuDBT chloride in full scan MS and MS/MS are reported for the first time using LC-APCI-MS. Gas chromatography-mass spectrometry (GC-MS) spectrum of the same compound is also reported for the first time for comparison purpose. This method allowed limits of detection (LOD) of 35 and 26 ng mL⁻¹ for TBT and OHBuDBT, respectively, based on successive injections of 10 μL of blank seawater extract. A liquid-liquid extraction procedure using n-hexane-ethyl acetate was developed for the simultaneous analysis of TBT and OHBuDBT chlorides in natural seawater and allowed average recoveries from 72 to 96% for the two compounds at three different spiking levels.     

Rapid detection of ketamine and norketamine in rat hair using micropulverized extraction and ultra‐performance liquid chromatography–electrospray ionization mass spectrometry

A new method for the rapid and simultaneous detection of ketamine and its major metabolite, norketamine, in rat hair has been developed by combining micropulverized extraction and ultraperformance liquid chromatography–electrospray ionization mass spectrometry. By using reversed‐phase UPLC, ketamine and norketamine were well separated within 2 min. Using ketamine‐dosed rat hair, the conditions for micropulverized extraction were optimized, and the limits of detection and quantification of the developed method were found to be 1.7 and 5.7 pg/mg hair for ketamine, respectively. The precisions achieved with this method were slightly better than that obtained with conventional acidic methanol extraction method. Using this proposed method, analysis of the washed rat hair could be completed within 16–17 min. This method is expected to be applied for the analysis of the hair samples of not only rats but also ketamine abusers. Copyright © 2009 John Wiley & Sons, Ltd.