Takeshi Kumazawa

Quantitative determination of phenothiazine derivatives in human plasma using monolithic silica solid-phase extraction tips and gas chromatography–mass spectrometry

Solid-phase extraction (SPE) using micropipette tips is a useful technique to prepare samples prior to mass spectrometry. However, most commercial SPE tips have loading capacities that are insufficient for quantitative determination. In this paper, we describe a rapid method for quantitative microanalysis of five phenothiazine derivatives, chlorpromazine, levomepromazine, promazine, promethazine and trimeprazine, using a recently introduced C(18) monolithic silica SPE tip, the MonoTip C(18), for extraction from human plasma. The drugs could be extracted within 5 min from 0.1-mL plasma samples, eluted with methanol, and the eluate injected directly into a gas chromatograph prior to mass spectrometry analysis. Only 0.7 mL of solvent was required for each step of the extraction process. The recoveries of the five phenothiazines spiked into plasma were 91-95% and the limits of quantification for each drug were between 0.25 and 2.0 ng/0.1 mL. The maximum intra- and inter-day coefficient of variation was 11%. The validated method was successfully used to quantify the plasma concentration of levemepromazine in a human subject after oral administration of the drug. This new method is expected to have wide applications as a pretreatment for the rapid, quantitative determination of drug concentrations in plasma samples.

New and unique methods of solid-phase extraction for use before instrumental analysis of xenobiotics in human specimens

For forensic toxicological analysis of human specimens, extractive pretreatments are critical for successful analysis of target compounds. Nowadays, solidphase extraction (SPE) cartridges or columns packed with silica-type or polymer-type spherical particles are most widely used. In recent years, monolithic sorbent products, which have sponge-like structures, have been developed for SPE. The most significant advantage of the monolithic sorbent is that it allows very fast flow through the sorbent structure. Utilizing this property, pipette tips and spin columns packed with the monolithic sorbent have become commercially available. In this review, we present some details of SPE techniques using the monolithic pipette tips and spin columns, and discuss their advantages. The recent development of molecularly imprinted polymers (MIPs), which can selectively trap target compounds and act as an SPE sorbent, is also discussed with consideration of their advantages and disadvantages in SPE.

High-throughput determination of nonsteroidal anti-inflammatory drugs in human plasma by HILIC-MS/MS

A simple and sensitive method was developed and validated here for the analysis of thirteen nonsteroidal anti-inflammatory drugs (NSAIDs) in human plasma samples by hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry (MS/MS). A small volume of plasma (20μL) spiked with compounds was diluted with 80μL of 10-mM ammonium acetate followed by a simple protein precipitation with 400μL of acetonitrile. After centrifugation, the clear supernatant extract was directly injected into the HILIC-MS/MS, without any solvent evaporation and reconstitution steps. The chromatographic separation of the NSAIDs was achieved on a Unison UK-Amino HILIC column (50mm×3mm i.d., particle size 3μm) with a linear gradient elution system composed of 10mM ammonium acetate (pH 6.8) and acetonitrile at a flow rate of 0.4mL/min. The mass spectra obtained by HILIC-MS showed base peak ions due to [M+H](+) for indomethacin, oxaprozin, ketoprofen, alminoprofen, zaltoprofen, tiaprofenic acid, pranoprofen, and ketoprofen-d3 and due to [M-H](-) for etodolac, ibuprofen, diclofenac, fenoprofen, loxoprofen, naproxen, and ibuprofen-d3. Recoveries of these thirteen NSAIDs in plasma were 34.8-113% and the lower limits of quantitation were 0.125-1.25μg/mL. The intra- and interday coefficient of variations for all drugs in plasma were less than 14.6%. The data obtained from actual plasma determinations of zaltoprofen, ibuprofen, and diclofenac are also presented.

High-throughput determination of theophylline and caffeine in human serum by conventional liquid chromatography-mass spectrometry

Automated high-performance liquid chromatography/mass spectrometry (HPLC-MS) with backflush column-switching was established for ultra-fast determination of theophylline and caffeine. A 400-μl portion of serum sample diluted with ultrapure water was injected and transferred to an Oasis HLB cartridge used as a precolumn for extraction. After switching the valves, the analytes trapped in the precolumn were eluted in the backflush mode and separated with a Chromolith Performance RP-18e column (C18-bonded monolithic silica); the compounds in column effluents were then detected by atmospheric pressure chemical ionization (APCI)-MS. The present method successfully provided high-throughput determination of theophylline and caffeine within 2 min. Satisfactory linearity, reproducibility, and sensitivity could be obtained for analysis of therapeutic and toxic levels of both compounds. Because of the very simple procedure and high throughput using the conventional HPLC system, the present method seems to have high potential in the fields of forensic toxicology and emergency medicine.

Automated on-line in-tube solid-phase microextraction coupled with HPLC/MS/MS for the determination of butyrophenone derivatives in human plasma

This paper describes a fully automated on-line method combining in-tube solid-phase microextraction (SPME) in which sample clean-up and enrichment are conducted through an open tubular fused-silica capillary column and high-performance liquid chromatography (HPLC)/tandem mass spectrometry (MS/MS) detection for the determination of six butyrophenone derivatives (moperone, floropipamide, haloperidol, spiroperidol, bromperidol, and pimozide) in human plasma samples. The six butyrophenones were extracted by repeatedly aspirating and dispensing plasma sample solutions on a DB-17 capillary column (60xa0cmu2009×u20090.32xa0mm i.d., film thickness 0.25xa0µm). The analytes retained on the inner surface of the capillary column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. Extraction efficiencies ranged from 12.7% to 31.8% for moperone, spiroperidol, and pimozide, and from 1.08% to 4.86% for floropipamide, haloperidol, and bromperidol. The regression equations for all compounds showed excellent linearity, ranging from 0.05 to 50xa0ng/0.1xa0mL of plasma, except for moperone and spiroperidol (0.01 to 50xa0ng/0.1xa0mL). The limits of detection and quantification in plasma for each drug were 0.03–0.2 and 0.1–0.5xa0ng/mL, respectively. The intra- and inter-day coefficients of variation for all compounds in plasma were not greater than 13.7%.

Determination of dextromethorphan in human plasma using pipette tip solid-phase extraction and gas chromatography-mass spectrometry.

Dextromethorphan was extracted from human plasma samples (100xa0μL) using MonoTip C18 tips, which are packed with C18-bonded monolithic silica gel that is attached to the inside of the tip. The samples, which contained dextromethorphan and trimeprazine as an internal standard (IS), were mixed with 200xa0μL of distilled water and 50xa0μL of 1xa0mol/L glycine–sodium hydroxide buffer (pHxa010). The mixture was extracted to the C18 phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C18 phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30xa0mu2009×u20090.32xa0mm i.d., film thickness 0.5xa0μm) gave adequate separation of the dextromethorphan, IS, and impurities. The recoveries of dextromethorphan and the IS spiked into plasma were >87.4%. The regression equation for dextromethorphan showed excellent linearity from 2.5 to 320xa0ng/mL of plasma, and the limit of detection was 1.25xa0ng/mL of plasma. The intraday and interday coefficients of variation were less than 10.5% and 14.7%, respectively. The accuracy ranged from 91.9% to 107%. The validated method was successfully used to quantify the plasma concentration of dextromethorphan in a human subject after oral administration of the drug.

Molecularly imprinted solid‐phase extraction for the selective determination of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs in human whole blood by gas chromatography‐mass spectrometry

A novel method is described for the extraction of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs, such as 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine, and 3,4-(methylenedioxyphenyl)-2-butanamine, from human whole blood using molecularly imprinted solid-phase extraction as highly selective sample clean-up technique. Whole blood samples were diluted with 10 mmol/L ammonium acetate (pH 8.6) and applied to a SupelMIP-Amphetamine molecularly imprinted solid-phase extraction cartridge. The cartridge was then washed to eliminate interferences, and the amphetamines of interest were eluted with formic acid/methanol (1:100, v/v). After derivatization with trifluoroacetic anhydride, the analytes were quantified using gas chromatography-mass spectrometry. Recoveries of the seven amphetamines spiked into whole blood were 89.1-102%. The limits of quantification for each compound in 200 μL of whole blood were between 0.25 and 1.0 ng. The maximum intra- and inter-day coefficients of variation were 9.96 and 13.8%, respectively. The results show that methamphetamine, amphetamine, and methylenedioxyphenylalkyl-amine designer drugs can be efficiently extracted from crude biological samples such as whole blood by molecularly imprinted solid-phase extraction with good reproducibility. This extraction method will be useful for the pretreatment of human samples before gas chromatography-mass spectrometry.

Activation of NFAT signal by p53-K120R mutant

The tumor suppressor p53 is activated by phosphorylation and/or acetylation. We constructed 14 non‐phosphorylated, 11 phospho‐mimetic, and 1 non‐acetylated point p53 mutations and compared their transactivation ability in U‐87 human glioblastoma cells by the luciferase reporter assay. Despite mutations at the phosphorylation sites, only the p53‐K120R and p53‐S9E mutants had marginally reduced activities. On the other hand, the Nuclear factor of activated T‐cells (NFAT)‐luciferase reporter was more potently activated by p53‐K120R than by wild‐type p53 and other mutants in glioblastoma, hepatoma and esophageal carcinoma cells. This suggests that acetylation at Lys‐120 of p53 negatively regulates a signaling pathway leading to NFAT activation.

A new method for quantitative determination of dimemorfan in human plasma using monolithic silica solid-phase extraction tips

Dimemorfan was extracted from human plasma samples (100 μL) using MonoTip C(18) tips, which were packed with a C(18)-bonded monolithic silica gel attached to the inside of the tip. The samples, which contained dimemorfan and trimeprazine as an internal standard (IS), were mixed with 300 μL of distilled water and 50 μL of 1M glycine-sodium hydroxide buffer (pH 10). The mixture was extracted onto the C(18) phase of the tip by 20 sequential aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five sequential aspirating/dispensing cycles. The eluate was injected directly into a gas chromatograph and detected by a mass spectrometer with selected ion monitoring in positive electron ionization mode. An Equity-5 fused silica capillary column (30 m × 0.32 mm i.d., film thickness 0.25 μm) gave adequate separation of the dimemorfan, IS, and impurities. The recoveries of dimemorfan and the IS spiked into plasma were ≥83%. The regression equation for dimemorfan showed excellent linearity from 0.25 to 32.0 ng/100 μL of plasma, and the limit of detection was 0.125 ng/100 μL of plasma. The maximum intra-day and inter-day relative standard deviations were 13%, while accuracy ranged from 88% to 105%. Dimemorfan was stable for at least 12 h at 4°C, 4 weeks at -80°C, and three freeze-thaw cycles in plasma. This new method is expected to have application as a pretreatment for the rapid, simple, and quantitative determination of dimemorfan in plasma samples.

Utility of disk solid-phase extraction for whole blood samples: analysis of some tetracyclic antidepressants by gas chromatography with nitrogen-phosphorus detection

Four tetracyclic antidepressants, maprotiline, mianserin, mirtazapine, and setiptiline, were extracted from human whole blood and plasma samples by disk solid-phase extraction with Empore C18 cartridges. They were determined by gas chromatography (GC) with nitrogen-phosphorus detection. Recoveries of maprotiline, mianserin, mirtazapine, and setiptiline spiked into whole blood or plasma were more than 83%. Regression equations for the four drugs showed excellent linearity in the range of 25–1000 ng in 0.2 ml whole blood and in 0.5 ml plasma. The limits of detection for the drugs were 4.1–18.2 ng per 0.2 ml for whole blood and 3.4–13.5 ng per 0.5 ml for plasma. The limits of quantification for the four drugs were 25 ng in 0.2 ml whole blood and in 0.5 ml plasma. Intraday and interday coefficients of variation for the antidepressants in both human specimens were below 15%. The above data show that relatively hydrophobic and basic drugs in crude biological samples such as whole blood can be extracted by disk solid-phase extraction very efficiently and with good reproducibility, and suggest that the extraction method is also useful as pretreatments before more sophisticated detection methods such as GC-mass spectrometry (MS) and liquid chromatography-MS(-MS).