Akemi Marumo

Pipette tip solid-phase extraction and gas chromatography-mass spectrometry for the determination of mequitazine in human plasma.

Mequitazine has been found to be extractable from human plasma samples using MonoTip C(18) tips, inside which C(18)-bonded monolithic silica gel was fixed. Human plasma (0.1mL) containing mequitazine and cyproheptadine as an internal standard (IS) was mixed with 0.4mL of distilled water and 25muL of 1M potassium phosphate buffer (pH 8.0). After centrifugation of the mixture, the supernatant fraction was extracted to the C(18) phase of the tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C(18) phase were then eluted with methanol by five repeated aspirating/dispensing cycles. Without evaporation and reconstitution, the eluate was injected into a gas chromatograph injector and detected by a mass spectrometer with selected ion monitoring in the positive-ion electron impact mode. The separation of mequitazine and the IS from each other and from impurities was generally satisfactory using a DB-1MS capillary column (30mx0.32mm i.d., film thickness 0.25mum). The recoveries of mequitazine and the IS spiked into plasma were more than 90.0%. The regression equation for mequitazine showed excellent linearity in the range of 0.2-200ng0.1mL(-1), and the detection limit was 0.05ng0.1mL(-1)of plasma. The intra-day and inter-day coefficients of variation for mequitazine in human plasma were not greater than 8.16 and 9.24%, respectively. Accuracy for the drug was in the range of 90.0-97.4%. The data obtained from determination of mequitazine in human plasma after oral administration of the drug are also presented.

Determination of diazepam and its metabolites in human urine by liquid chromatography/tandem mass spectrometry using a hydrophilic polymer column

Diazepam and its major metabolites, nordazepam, temazepam and oxazepam, in human urine samples, were analyzed by liquid chromatography (LC)/tandem mass spectrometry (MS/MS) using a hydrophilic polymer column (MSpak GF-310 4B), which enables direct injection of crude biological samples. Matrix compounds in urine were eluted first from the column, while the target compounds were retained on the polymer stationary phase. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All compounds showed base-peak ions due to [M+H]+ ions on LC/MS with positive ion electrospray ionization, and product ions were produced from each [M+H]+ ion by LC/MS/MS. Quantification was performed by selected reaction monitoring. All compounds spiked into urine showed method recoveries of 50.1-82.0%. The regression equations for all compounds showed excellent linearity in the range of 0.5-500 ng/mL of urine. The limits of detection and quantification for each compound were 0.1 and 0.5 ng/mL of urine, respectively. The intra- and inter-day coefficients of variation for all compounds in urine were not greater than 9.6%. The data obtained from actual determination of diazepam and its three metabolites, oxazepam, nordazepam and temazepam, in human urine after oral administration of diazepam, are also presented.

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.

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).

Extraction of Muscle Relaxants in Human Body Fluids by Solid‐Phase Extraction

Abstract Three muscle relaxants, tolperisone, eperisone, and tizanidine, were found to be extractable from human whole blood and urine samples by solid‐phase extraction (SPE) with a Sep‐Pak C18 cartridge. Their determination was made using capillary gas chromatography with nitrogen phosphorus detection. Recoveries of the three drugs were 80.0%–97.2% for whole blood and 82.0%–94.5% for urine. The coefficients of within‐day variation in terms of recovery for all compounds in whole blood and urine samples were 2.2%–9.1% and 6.9%–9.6%, respectively; those of day‐to‐day variation for the compounds in both samples were not greater than 18.1%. The regression equations for the compounds showed excellent linearity with detection limits of 1.6–7.7 ng mL−1 for whole blood and 6.0–8.0 ng mL−1 for urine. The data obtained for actual determination of tolperisone in a subject after ingestion of the drugs are also presented.

SPIN TIP SOLID-PHASE EXTRACTION AND HILIC-MS-MS FOR QUANTITATIVE DETERMINATION OF METHAMPHETAMINE AND AMPHETAMINE IN HUMAN PLASMA

A rapid and simple microanalysis method using TopTip C18 spin tips, which are new micropipette tip spin columns for solid-phase extraction, was developed and validated for quantitative determination of methamphetamine and amphetamine in human plasma. All the solid-phase extraction procedures, including conditioning, sample loading, washing, and elution, using the TopTip C18 spin tips, were performed by centrifugation. The analytes were extracted within 7–8 min from 0.1 mL of plasma. The methanol-rich eluate was injected directly into a hydrophilic interaction liquid chromatography/tandem mass spectrometer in positive electrospray ionization mode with selected reaction monitoring. The analytes were separated on an Imtakt Unison UK-silica column using a gradient with methanol and 10 mmol/L ammonium acetate buffer as mobile phase at a flow rate of 0.5 mL/min. The recoveries of methamphetamine and amphetamine spiked into plasma were 82–95% and the limit of quantification of methamphetamine and amphetamine were 2.5 ng/mL of plasma. The calibration curves were linear over the concentration range of 2.5–500 ng/mL of plasma for both compounds. The intra-day and inter-day relative standard deviations were less than 7.5%. This fast and simple method is expected to be very useful for quantitative determination of methamphetamine and amphetamine in human plasma samples.