Zheng Ouyang

Systematic LC‐MS/MS bioanalytical method development that incorporates plasma phospholipids risk avoidance, usage of incurred sample and well thought‐out chromatography

This treatise summarizes the underlying principle and the road map for systematic LC-MS/MS bioanalytical method development. The three themes that have recently emerged as central to building quality during method development-phospholipids, incurred sample and sound chromatographic considerations-are the main focus of this article. In order to reduce the bioanalytical risk associated with plasma phospholipids, a dual approach involving extraction and chromatography is recommended. The use of incurred sample during method development is essential to avoid interference arising from drug-related components. This is different from the current practice of incurred sample reanalysis, which tests reproducibility during method application. LC column/mobile phase optimization is needed to achieve appropriate peak shape, sensitivity and the separation of the analyte from interfering metabolites and phospholipids. Related to sound chromatographic considerations, we lay out facts and myths related to UPLC, vis-à-vis HPLC. Incorporation of quality during method development avoids the costly experience of finding out by chance about the invalidity of a method after it has been used in support of a number of pivotal clinical and non-clinical studies. To this end, we put forth an outline of a protocol for a systematic LC-MS/MS bioanalytical method development.

Quantitation of the acid and lactone forms of atorvastatin and its biotransformation products in human serum by high-performance liquid chromatography with electrospray tandem mass spectrometry

A method for simultaneous quantitation of both the acid and lactone forms of atorvastatin, a new synthetic inhibitor of HMG-CoA reductase that is being marketed for the treatment of high serum cholesterol, and both the acid and lactone forms of its two biotransformation products, 2-hydroxyatorvastatin and 4-hydroxyatorvastatin, in human serum (a total of six analytes) by high-performance liquid chromatography with electrospray tandem mass spectrometry was developed and validated. A deuterium labeled analog was used as internal standard for each of the six analytes. Each point of the calibration standard curve, which ranged from 0.5 to 200 ng/mL, contained the six analytes at equal concentrations. Three groups of quality control (QC) samples were used. In the first group, combination QC samples contained all six analytes at equal concentrations. In the second group, acid-only QC samples contained only the acid forms (i.e. three analytes) at equal concentrations. In the third group, lactone-only QC samples contained only the lactone forms (i.e. three analytes) at equal concentrations. After adding the internal standard to 0.5 mL of each standard and the QC sample kept at 4 degrees C, the samples were acidified with sodium acetate buffer (pH 5.0) and then extracted with methyl tert-butyl ether. Detection was by positive ion electrospray tandem mass spectrometry using eight selected reaction monitoring channels. The acid compounds were stable in human serum at room temperature but the lactone compounds were unstable as they hydrolyzed rapidly to their respective acid forms. The conversion of the lactone compounds in both QC and post-dose human serum samples was nearly complete after 24 h at room temperature. The lactone compounds in serum could be stabilized by lowering the working temperature to 4 degrees C or lowering the serum pH to 6.0. The acid-only and the lactone-only QC samples showed that, under the sample processing conditions used, the degree of the hydrolysis of the lactone compounds or the lactonization of the acid compounds during the assay procedure was minimal (< 5%). The intra-day C.V., inter-day C.V. and the deviations from the nominal concentrations for all six analytes were within 15%, demonstrating good precision and accuracy. The required lower limit of quantitation (LLQ) of 0.5 ng/mL was achieved for each analyte.

Direct-injection LC–MS–MS method for high-throughput simultaneous quantitation of simvastatin and simvastatin acid in human plasma

A direct-injection liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) method was developed and validated for the simultaneous quantitation in human plasma of the widely used cholesterol-lowering prodrug simvastatin and its in vivo generated active drug, simvastatin acid. The plasma samples were injected into the LC-MS-MS system after simply adding the internal standard solution in an aqueous buffer and centrifuging. The analytes in the buffered plasma samples were found to be stable for at least 24 h at 4 degrees C. The method was successfully validated under the challenging condition of using a large number of quality control (QC) samples including those in which the ratio of the simvastatin concentration to the simvastatin acid concentration was different from the concentration ratio in the calibration curve standards. Under the dual stabilizing conditions of lower temperature (4 degrees C) and lower plasma pH of 4.9, the in-process hydrolysis of simvastatin to simvastatin acid or the lactonization of simvastatin acid to simvastatin was minimized to < or = 1.0%. Although the entire run time for on-line cleanup and analysis was only 2.5 min, chromatographic base-line separation of simvastatin from simvastatin acid, which was required to avoid the interference by simvastatin acid with the simvastatin selected reaction monitoring channel, was achieved. The desired lower limit of quantitation of 0.5 ng/ml was achieved by injecting only an equivalent of 8.0 microl of the plasma sample. The extraction column lasted for at least 500 injections.

Pellet digestion: a simple and efficient sample preparation technique for LC–MS/MS quantification of large therapeutic proteins in plasma

BACKGROUND There is a need for a simple and efficient sample preparation technique for LC-MS/MS quantification of large therapeutic proteins in plasma. RESULTS The sample preparation technique presented here is based upon trypsin digestion of the pellet obtained following precipitation of the protein analyte from plasma. The pellet digestion technique was shown to facilitate efficient digestion of large therapeutic proteins, with concomitant removal of a substantial amount of potentially problematic plasma phospholipids. The technique was successfully applied to a pharmacokinetic study of a large therapeutic protein. CONCLUSION This simple sample preparation approach will be beneficial to bioanalytical laboratories engaged in the LC-MS/MS quantification of large therapeutic proteins in biological matrices.

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS-MS). The first procedure was based on the manual liquid-liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.

A strategy for liquid chromatography/tandem mass spectrometry based quantitation of pegylated protein drugs in plasma using plasma protein precipitation with water‐miscible organic solvents and subsequent trypsin digestion to generate surrogate peptides for detection

Recently, we have developed liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based methods for the quantitation of pegylated therapeutic proteins in plasma. The methods are based on the LC/MS/MS detection of a surrogate peptide generated from trypsin digestion of the therapeutic protein. Various parameters related to the bioanalytical methods were evaluated and optimized, including the preparation of calibration standards and quality control samples, sample extraction, internal standard selection and its stage of addition, trypsin digestion, and non-specific binding. In this paper, we report the development of a method for a specific pegylated therapeutic protein and detail the various optimization steps undertaken. Simple extraction of the pegylated therapeutic protein from plasma was achieved via the precipitation of the endogenous proteins in plasma using acidic isopropanol and the resulting supernatant extract was subjected to trypsin digestion. A unique tryptic peptide arising from the pegylated therapeutic protein was used for LC/MS/MS-based detection and quantitation. A protein and a peptide were used as internal standards, with the former added before the sample extraction and the latter after the sample extraction. The method developed is simple, sensitive, specific and rugged, and has been implemented in a high throughput 96-well format to analyze plasma samples from in vivo studies. A required lower limit of quantitation (LLOQ) of 10 ng/mL, expressed in terms of the concentration of the protein drug, was easily achieved.

The need for chromatographic and mass resolution in liquid chromatography/tandem mass spectrometric methods used for quantitation of lactones and corresponding hydroxy acids in biological samples

Because of the potential in-source conversion between a lactone and the corresponding hydroxy acid, it has been recognized that a liquid chromatography/tandem mass spectrometric (LC/MS/MS) method developed for quantitation of a lactone drug in the presence of its hydroxy acid metabolite (or vice versa) must incorporate chromatographic separation between the two compounds, unless in-source conversion between the two compounds has been eliminated by the appropriate selection of the LC/MS/MS parameters. We now report that chromatographic separation between a lactone and its hydroxy acid will be required under certain LC/MS/MS conditions used even in the absence of in-source conversion. This is due to the fact that the 18-mass-unit difference between a lactone and its hydroxy acid is, by coincidence, different by only one mass unit from the 17-mass-unit difference between the [M + H](+) and [M + NH(4)](+) ions of the lactone or the hydroxy acid. Thus, the [M + H](+) ion of a hydroxy acid is higher than the [M + NH(4)](+) ion of its lactone by only one mass unit. Therefore, in a method developed for quantitation of a hydroxy acid drug utilizing a selected-ion-monitoring (SRM) scheme that incorporates its [M + H](+) ion as the precursor ion, the quantitation would be inaccurate due to the interference by the contribution of the A + 1 isotope response from the [M + NH(4)](+) ion of the lactone metabolite present in the sample, unless there is a chromatographic separation between the two compounds. This is true even if Q1 is operated under a unit-mass resolution. The implication of this type of interference, arising from the presence of both the [M + H](+) and [M + NH(4)](+) ions of a drug and its metabolite, to the selection of LC and MS conditions (including mass resolution) will be discussed using the data obtained with a model lactone drug and its hydroxy acid metabolite.

A versatile system of high‐flow high performance liquid chromatography with tandem mass spectrometry for rapid direct‐injection analysis of plasma samples for quantitation of a β‐lactam drug candidate and its open‐ring biotransformation product

A bioanalytical method has been developed and validated for quantitation of a beta-lactam drug candidate and its open-ring biotransformation product utilizing high-flow high-performance liquid chromatography (HPLC) for on-line purification of plasma samples and electrospray tandem mass spectrometry for detection and quantitation. The HPLC system used two columns: an Oasis column (1 x 50 mm, 30 microm) as the on-line extraction column and a conventional C18 column (2 x 50 mm, 5 microm) as the analytical column. Each plasma standard or quality control (QC) sample (50 microL) was mixed with 50 microL of a working solution of the internal standard in aqueous 0.5 M ammonium acetate (pH 4.0). Portions (10 microL) of these samples were then injected into an Oasis column with a mobile phase consisting of 100% aqueous 1 mM formic acid at a high flow rate (4.0 mL/min), with the effluent from the Oasis column directed to waste and not to the mass spectrometer. After the purification step, the Oasis column effluent was directed to the analytical column and the mass spectrometer and the analytes were eluted with methanol/aqueous 1 mM formic acid (70:30) at a flow rate of 1.0 mL/min. The total analysis time was 1.6 min per sample. The standard curve range was 0.980 to 250 ng/mL. The accuracy, inter-day precision and intra-day precision were within 10% for both compounds.

A User-Friendly Robotic Sample Preparation Program for Fully Automated Biological Sample Pipetting and Dilution to Benefit the Regulated Bioanalysis

Biological sample dilution is a rate-limiting step in bioanalytical sample preparation when the concentrations of samples are beyond standard curve ranges, especially when multiple dilution factors are needed in an analytical run. We have developed and validated a Microsoft Excel–based robotic sample preparation program (RSPP) that automatically transforms Watson worklist sample information (identification, sequence and dilution factor) to comma-separated value (CSV) files. The Freedom EVO liquid handler software imports and transforms the CSV files to executable worklists (.gwl files), allowing the robot to perform sample dilutions at variable dilution factors. The dynamic dilution range is 1- to 1000-fold and divided into three dilution steps: 1- to 10-, 11- to 100-, and 101- to 1000-fold. The whole process, including pipetting samples, diluting samples, and adding internal standard(s), is accomplished within 1 h for two racks of samples (96 samples/rack). This platform also supports online sample extraction (liquid-liquid extraction, solid-phase extraction, protein precipitation, etc.) using 96 multichannel arms. This fully automated and validated sample dilution and preparation process has been applied to several drug development programs. The results demonstrate that application of the RSPP for fully automated sample processing is efficient and rugged. The RSPP not only saved more than 50% of the time in sample pipetting and dilution but also reduced human errors. The generated bioanalytical data are accurate and precise; therefore, this application can be used in regulated bioanalysis.

A rugged and accurate liquid chromatography–tandem mass spectrometry method for the determination of asunaprevir, an NS3 protease inhibitor, in plasma

Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) non-structural protein protease inhibitor currently in Phase III clinical trials for the treatment of HCV infection. A rugged and accurate LC-MS/MS method was developed and validated for the quantitation of asunaprevir in rat, dog, monkey, rabbit and mouse plasma. A systematic method screening and optimization strategy was applied to achieve optimized mass spectrometry, chromatography, and sample extraction conditions. The validated method utilized stable-isotope labeled D9-asunaprevir as the internal standard. The samples were extracted by liquid-liquid extraction using 10% ethyl acetate in hexane. Chromatographic separation was achieved with gradient elution on a Waters Atlantis dC18 analytical column. Analyte and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The standard curve, which ranged from 5.00 to 2000ng/mL for asunaprevir, was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within ±3.6% CV, inter-assay precision was within ±4.0% CV, and the assay accuracy was within ±8.1% of the nominal values in all the species. The method was successfully applied to support multiple pre-clinical toxicokinetic studies in different species.