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.

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.

Pipette tip solid-phase extraction and gas chromatography - mass spectrometry for the determination of methamphetamine and amphetamine in human whole blood.

Methamphetamine and amphetamine were extracted from human whole blood samples using pipette tip solid-phase extraction (SPE) with MonoTip C(18) tips, on which C(18)-bonded monolithic silica gel was fixed. Human whole blood (0.1 mL) containing methamphetamine and amphetamine, with N-methylbenzylamine as an internal standard, was mixed with 0.4 mL of distilled water and 50 microL of 5 M sodium hydroxide solution. After centrifugation, the supernatant was extracted to the C(18) phase of the tip (pipette tip volume, 200 microL) by 25 repeated aspirating/dispensing cycles using a manual micropipettor. Analytes retained in the C(18) phase were eluted with methanol by five repeated aspirating/dispensing cycles. After derivatization with trifluoroacetic anhydride, analytes were measured by gas chromatography - mass spectrometry with selected ion monitoring in the positive-ion electron impact mode. Recoveries of methamphetamine and amphetamine spiked into whole blood were more than 87.6 and 81.7%, respectively. Regression equations for methamphetamine and amphetamine showed excellent linearity in the range of 0.5-100 ng/0.1 mL. The limits of detection for methamphetamine and amphetamine were 0.15 and 0.11 ng/0.1 mL, respectively. Intra- and interday coefficients of variation for both stimulants were not greater than 9.6 and 13.8%, respectively. The determination of methamphetamine and amphetamine in autopsy whole blood samples is presented, and was shown to validate the present methodology.

Simultaneous determination of ten antihistamine drugs in human plasma using pipette tip solid-phase extraction and gas chromatography/mass spectrometry.

Ten antihistamine drugs, diphenhydramine, orphenadrine, chlorpheniramine, diphenylpyraline, triprolidine, promethazine, homochlorcyclizine, cyproheptadine, cloperastine and clemastine, have been found to be extractable from human plasma samples using MonoTip C18 tips, inside which C18- bonded monolithic silica gel was fixed. Human plasma (0.1 mL) containing the ten antihistamines was mixed with 0.4 mL of distilled water and 25 microL of a 1 M potassium phosphate buffer (pH 8.0). After centrifugation of the mixture, the supernatant fraction was extracted to the C18 phase of the tip by 25 repeated aspirating/dispensing cycles using a manual micropipettor. The analytes retained on the C18 phase were then eluted with methanol by five repeated aspirating/dispensing cycles. The eluate was injected into a gas chromatography (GC) injector without evaporation and reconstitution steps, and was detected by a mass spectrometer with selected ion monitoring in the positive-ion electron impact mode. The separation of the ten drugs from each other and from impurities was generally satisfactory using a DB-1MS column (30 m x 0.32 mm i.d., film thickness 0.25 microm). The recoveries of the ten antihistamines spiked into plasma were 73.8-105%. The regression equations for the ten antihistamines showed excellent linearity with detection limits of 0.02-5.0 ng/0.1 mL. The within-day and day-to-day coefficients of variation for plasma were not greater than 9.9%. The data obtained from determination of diphenhydramine and chlorpheniramine in human plasma after oral administration of the drugs are also presented.

Simple method for determination of triazolam in human plasma by high-performance liquid chromatography/tandem mass spectrometry.

Triazolam was analyzed from human plasma samples by high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) with an MSpak GF polymer column (50 mm x 4.6 mm i.d., particle size 6 microm), which enabled direct injection of crude biological samples. Separation of triazolam, and lorazepam as the internal standard (IS) was carried out using 10mM ammonium acetate (pH 3.56)-0.1% formic acid and an acetonitrile gradient elution. Both compounds formed base peaks due to [M + H]+ ions by HPLC/ESI-MS, and product ions were produced from each [M + H]+ ion as seen by HPLC-MS/MS. Quantification of triazolam and the IS in plasma samples was made by selective reaction monitoring using each base peak of product ions of HPLC-MS/MS. The recovery range of triazolam spiked into plasma was 86.4-92.7%. The regression equation for triazolam showed excellent linearity in the range of 0.25-20 ng/mL, and the detection limit was 0.1 ng/mL. Intra- and inter-day precisions for triazolam in plasma samples were not greater than 12.4%. Accuracy for the drug was in the range of 88.0-101.4%. Data obtained after oral administration of triazolam in male and female subjects are also presented.

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.

Analysis of phenothiazines in human body fluids using disk solid-phase extraction and liquid chromatography.

Seven phenothiazine derivatives, perazine, perphenazine, prochlorperazine, propericiazine, thioproperazine, trifluoperazine, and flupentixol, have been found to be extractable from human plasma and urine samples using disk solid-phase extraction (SPE) with an Empore C18 cartridge. Human plasma and urine (1 mL each) containing the 7 phenothiazine derivatives were mixed with 2 mL of 0.1M NaOH and 7 mL distilled water and then poured into the disk SPE cartridges. The drugs were eluted with 1 mL chloroform- acetonitrile (8 + 2) and determined by liquid chromatography with ammonium formate/formic acid-acetonitrile gradient elution. The detection was performed by ultraviolet absorption at 250 nm. The separation of the 7 phenothiazine derivatives from each other and from impurities was generally satisfactory using a SymmetryShield RP8 column (150 x 2.1 mm id, 3.5 microm particle size). The recoveries of the 7 phenothiazine derivatives spiked into plasma and urine samples were 64.0-89.9% and 65.1-92.1%, respectively. Regression equations for the 7 phenothiazine derivatives showed excellent linearity, with detection limits of 0.021-0.30 microg/mL for plasma and 0.017-0.30 microg/mL for urine. The within-day and day-to-day coefficients of variation for both samples were commonly below 9.0 and 14.9%, respectively.