Xiangyu Jiang

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 assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase.

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC-MS-MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC-MS-MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC-MS interface. The standard curve range in plasma was 0.5-50 ng/ml for MOR, 1.0-100 ng/ml for M6G, and 10-1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC-MS-MS systems to analyze over one thousand samples from clinical trials.

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.

Development and validation of a sensitive and robust LC-tandem MS method for the analysis of warfarin enantiomers in human plasma.

A liquid chromatography-tandem mass spectrometry method (LC-MS-MS) was developed and validated for measuring warfarin (WAR) enantiomers (R-WAR and S-WAR) in human EDTA plasma. Liquid-liquid extraction using ethyl ether was used to extract the analytes from the plasma. Baseline resolution of S- and R-WAR as well as the internal standard enantiomers (S- and R-p-ClWAR, S-IS and R-IS) was achieved on a beta-cyclodextrin column with a mobile phase of acetonitrile-acetic acid-triethylamine (1000:3:2.5, v/v/v). The retention times are 6.9, 8.0, 7.0, and 7.9 min for S-WAR, R-WAR, S-IS and R-IS, respectively. The detection was by monitoring S- and R-WAR at m/z 307-->161 and S- and R-IS at m/z 341-->161 using (-) ESI. The standard curve range was 1-100 ng ml(-1) for both S- and R-WAR. The inter-day precision and accuracy of the quality control (QC) samples were <7.3% relative standard deviation (RSD) and <7.3% bias for S-WAR, and <6.5% RSD and <5.8% bias for R-WAR, respectively. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from analysis of clinical samples.

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.

Development and validation of a sensitive method for hydromorphone in human plasma by normal phase liquid chromatography–tandem mass spectrometry

Liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS) was developed for the quantitation of hydromorphone (HYD), an opiate analgesic, in human plasma. A simple liquid-liquid extraction was used to extract the analyte and its deuterated internal standard (d3-HYD). Chromatographic separation of hydromorphone from its metabolite hydromorphone-3-glucuronide (H3G) was necessary because of the significant H3G fragmentation to HYD before Ql of the mass spectrometer, which could result in false detection as HYD in the multiple reaction mode (MRM). This separation was achieved using a 50 x 2 mm, I.D. silica column (5 microm) and a mobile phase of acetonitrile-water formic acid (80:20:1, v/v/v). The method was validated in the concentration range 0.05-10 ng ml(-1) in plasma and met the acceptance criteria of industry guidelines for accuracy, precision, and stability.

Using supported liquid extraction together with cellobiohydrolase chiral stationary phases-based liquid chromatography with tandem mass spectrometry for enantioselective determination of acebutolol and its active metabolite diacetolol in spiked human plasma

A high through-put, sensitive, and enantioselective LC-MS/MS-based bioanalytical method was developed and validated for the simultaneous determination of individual acebutolol (AC) and its active metabolite-diacetolol (DC) enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP). Systematic optimization of chromatographic conditions including organic content, buffer concentration, and pH of mobile phases was conducted to improve the through-put for the direct separation of both AC and DC on CBH column during method development. Complete baseline separation of enantiomeric AC and DC was achieved within 1.5 min with a LC flow rate of 0.9 ml/min under method validation conditions. To further improve the assay through-put, supported liquid extraction (SLE) in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.0500-50.0 ng/ml for each AC and DC enantiomer using 0.100 ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed <or=4.5% relative standard deviation (R.S.D.) and -8.7 to 5.6% relative error (R.E.) for individual AC and DC enantiomers.

Simultaneous determination of hydrocodone and hydromorphone in human plasma by liquid chromatography with tandem mass spectrometric detection

A rapid, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the simultaneous analysis of hydrocodone (HYC) and its metabolite hydromorphone (HYM) in human plasma. A robotic liquid handler and a 96-channel liquid handling workstation were used to aliquot samples, to add internal standard (I.S.), and to extract analytes of interest. A 96-well mixed-mode solid-phase cartridge plate was used to extract the analytes and I.S. The chromatographic separation was on a silica column (50 x 3 mm, 5-microm) with a mobile phase consisting of acetonitrile, water and trifluoroacetic acid (TFA) (92:8:0.01, v/v). The run time for each injection was 2.5 min with the retention times of approximately 2.1 and 2.2 min for HYC and HYM, respectively. The tandem mass spectrometric detection was by monitoring singly charged precursor-->product ion transition 300-->199 (m/z) for HYC, and 28-->185 (m/z) for HYM. The validated calibration curve range was 0.100-100 ng/ml, based on a plasma volume of 0.3 ml. The correlation coefficients were greater than or equal to 0.9996 for both HYC and HYM. The low limit of quantitation (LLOQ) was 0.100 ng/ml for both HYC and HYM with signal-to-noise ratio (S/N) of 50 and 10. respectively. The deuterated analytes, used as internal standards, were monitored at mass transitions 303-->199 (m/z) for HYC-d3 and 289-->185 (m/z) for HYM-d3. The inter-day (n= 17) precision of the quality control (QC) samples were < or = 3.5% RSD (relative standard deviation) for HYC and < or = 4.7% RSD for HYM, respectively. The inter-day accuracy of the QC samples were < or = 2.1% RE (relative error) for HYC and < or = 1.8% RE for HYM. The intra-day (n=6) precision and accuracy of the QC samples were < or = 2.6% RSD and < or = 3.0% RE for HYC, and < or = 4.7% RSD and < or = 2.4% RE for HYM. There was no significant deviation from the nominal values after a 5-fold dilution of high concentration QC samples by blank matrix. The QC samples were stable when kept at room temperature for 24-h or experienced three freeze-thaw cycles. The extraction recoveries were 86% for HYC and 78% for HYM. No detectable carryover was observed when a blank sample was injected immediately after a 2500 ng/ml sample that was 25-fold more concentrated than the upper limit of quantitation (ULOQ).