Wilson Z. Shou

Novel liquid chromatographic-tandem mass spectrometric methods using silica columns and aqueous-organic mobile phases for quantitative analysis of polar ionic analytes in biological fluids.

Use of silica stationary phase and aqueous-organic mobile phases could significantly enhance LC-MS-MS method sensitivity. The LC conditions were compatible with MS detection. Analytes with basic functional groups were eluted with acidic mobile phases and detected by MS in the positive ion mode. Analytes with acid functional groups were eluted with mobile phases at neutral pH and detected by MS in the negative ion mode. Analytes poorly retained on reversed-phase columns showed good retention on silica columns. Compared with reversed-phase LC-MS-MS, 5-8-fold sensitivity increases were observed for basic polar ionic compounds when using silica columns and aqueous-organic mobile phase. Up to a 20-fold sensitivity increase was observed for acidic polar ionic compounds. Silica columns and aqueous-organic mobile phases were used for assaying nicotine, cotinine, and albuterol in biological fluids.

Simultaneous development of six LC-MS-MS methods for the determination of multiple analytes in human plasma

Traditional sequential single analyte method development is both time-consuming and labor-intensive. In this report, a concept of simultaneously developing multiple liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) methods were proposed. Mass spectrometric and chromatographic conditions as well as sample preparation methods for all analytes were optimized concurrently. Mass spectrometric conditions for six analytes, i.e. clonidine (CLO), albuterol (ALB), fentanyl (FEN), ritonavir (RIT), naltrexone (NAL), and loratadine (LOR), were established simultaneously using the Sciex Analyst software. LC-MS-MS sensitivities obtained using gradient elution methods on reversed-phase Inertsil ODS3 and normal phase Betasil silica columns were compared. Sample extraction methods using protein precipitation, liquid/liquid extraction, or solid-phase extraction (SPE) were evaluated. Recovery of analytes was determined. Matrix effects and interference due to endogenous compounds were investigated. Selection of a potential internal standard was discussed.

An automatic 96-well solid phase extraction and liquid chromatography-tandem mass spectrometry method for the analysis of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma.

A bioanalytical method using automated sample transferring, automated solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed for morphine (MOR), and its metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in human plasma. Samples of 0.25 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe II). Automated SPE was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using a C(18) sorbent. The extract was injected onto a silica column using an aqueous-organic mobile phase. The chromatographic run time was 3.5 min per injection, with retention times of 1.5, 2.0 and 2.6 min for MOR, M6G, and M3G, respectively. The detection was by monitoring MOR at m/z 286-->152, M6G and M3G at m/z 462-->286. The deuterated internal standards were monitored at m/z 289-->152 for MOR-d(3), and m/z 465-->289 for M6G-d(3) and M3G-d(3). The standard curve range was 0.5-50 ng ml(-1) for MOR, 1.0-100 ng ml(-1) for M6G, and 10-1000 ng ml(-1) for M3G. The inter-day precision and accuracy of the quality control samples were <8% relative standard deviation (RSD) and <7% relative error (RE) for MOR, <5% RSD and <2% RE for M6G, and <2% RSD and <4% RE for M3G.

Liquid/liquid extraction using 96-well plate format in conjunction with hydrophilic interaction liquid chromatography–tandem mass spectrometry method for the analysis of fluconazole in human plasma

A bioanalytical method using automated sample transferring, automated liquid/liquid extraction (LLE) and hydrophilic interaction liquid chromatography-tandem mass spectrometry was developed for the determination of fluconazole in human plasma. Samples of 0.05 ml were transferred into 96-well plate using automatic liquid handler (Multiprobe II). Automated LLE was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using methyl-tetra butyl ether as the extraction solvent. The extract was evaporated to dryness, reconstituted, and injected onto a silica column using an aqueous-organic mobile phase. The chromatographic run time was 2.0 min per injection, with retention times of 1.47 and 1.44 min for fluconazole and internal standard (IS) ritonavir, respectively. The detection was by monitoring fluconazole at m/z 307-->238 and IS at m/z 721-->296, respectively. The standard curve range was 0.5-100 ng ml(-1). The inter-day precision and accuracy of the quality control samples were <7.1% relative standard deviation and <2.2% relative error.

Development and validation of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of ribavirin in human plasma and serum

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of antiviral agent ribavirin in human plasma and serum. The samples (0.1 ml) were extracted from the matrix using a simple protein precipitation procedure. The supernatants were evaporated to dryness, reconstituted and injected onto the LC/MS/MS system. The chromatography separation was achieved on a silica column operated with an aqueous-organic mobile phase. The use of a silica column not only provided adequate retention for the extremely polar compound of ribavirin, but also enhanced electrospray ionization sensitivity with the use of high percentage organic solvent in the mobile phase. The method has been validated over the concentration range of 10-10000 ng/ml ribavirin in human plasma and serum. Bamethan was used as the internal standard. The protein precipitation extraction has been automated based on 96-well format with the use of robotic liquid handlers to improve the overall throughput of the analysis.

Importance of injection solution composition for LC-MS-MS methods.

For the first time, the influence of the injection solution composition on the quality of LC-MS-MS methods, in terms of column efficiency and peak shape, was systematically investigated. Various types of compounds, including polar ionic acidic, polar ionic basic and non-polar neutral compounds, were prepared in different solutions ranging from 100% water to 100% acetonitrile. Different volumes of these solutions were injected onto either C18 or silica columns connected to tandem mass spectrometry. The mobile phases consisted of acetonitrile, water, and small amounts of volatile acid or buffer. On silica columns, the influence of injection solution on the peak shape and column efficiency was straightforward. The sharpest peaks and the highest column efficiency were obtained with 100% acetonitrile as the injection solvent. On C18 columns, this type of influence was less clear due to the dual retention mechanism of the bonded phase and of the residual silanol groups. On C18 column, retention due to residual silanol groups was significant even with a mobile phase containing less than 50% acetonitrile. Poor peak shape was observed when the injection solution had a stronger eluting strength than mobile phase, particularly for early eluting peaks.

A SENSITIVE AND HIGH-THROUGHPUT LC/MS/MS METHOD USING A SILICA COLUMN AND AN AQUEOUS-ORGANIC MOBILE PHASE FOR THE ANALYSIS OF FLUOXETINE AND NORFLUOXETINE IN HUMAN PLASMA

ABSTRACT A sensitive and high-throughput LC/MS/MS method was developed and validated for the determination of fluoxetine and its metabolite norfluoxetine in human plasma. Analytes of interest were extracted from alkalized human plasma using liquid/liquid extraction. The extract was injected onto a silica column with an aqueous-organic mobile phase consisting of acetonitrile, water, trifluoroacetic acid (TFA), and ammonium acetate. The chromatographic run time was 2.0 min per injection, with retention time of 1.1 min for both fluoxetine and norfluoxetine, and 1.2 min for the internal standard (IS), fentanyl-d5. Fluoxetine was monitored at m/z 310→44, norfluoxetine at 296→134, and IS at m/z 342→188, respectively. The standard curve range was 0.5–250 ng/mL and low limit of quantitation (LLOQ) was 0.5 ng/mL for both fluoxetine and norfluoxetine. The inter-day precision and accuracy of the quality control (QC) samples were <5.1% relative standard deviation (RSD) and <7.3% relative error (RE).