Verapamil quantification in human plasma by liquid chromatography coupled to tandem mass spectrometry. An application for bioequivalence study

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of Verapamil in human plasma using Metoprolol as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction and chromatographed on a C(8) analytical column. The mobile phase consisted of methanol-water (70:30; v/v)+12 mM formic acid. The method had a chromatographic total run time of 3.5 min and was linear within the range 1.00-500 ng/mL. Detection was carried out on a Micromass Quattro Ultima tandem mass spectrometer by multiple reaction monitoring (MRM). The intra-run imprecision was less than 5.1% calculated from the quality control (QC) samples, and 16.3% from the limit of quantification (LOQ). The accuracy determined from QC samples were between 92.9 and 103.1%, and 95.2 and 115.3% from LOQ. Concerning the inter-batch analysis, the imprecision was less than 5.8% and 17.3% from QC samples and LOQ, respectively. The accuracy varied between 98.2 and 100.8% from QC and it was 103.1% from LOQ. The protocol herein described was employed in a bioequivalence study of two tablet formulations of Verapamil.     

Lansoprazole quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of lansoprazole in human plasma using omeprazole as the internal standard. The analyte and internal standard were extracted from the plasma samples by liquid-liquid extraction using diethyl-ether-dichloromethane (70:30; v/v) and chromatographed on a C(18) analytical column. The mobile phase consisted of acetonitrile-water (90:10; v/v)+10 mM formic acid. The method has a chromatographic total run time of 5 min and was linear within the range 2.5-2000 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by Multiple Reaction Monitoring (MRM). The intra- and inter-run precision, calculated from quality control (QC) samples, was less than 3.4%. The accuracy as determined from QC samples was less than 9%. The method herein described was employed in a bioequivalence study of two capsule formulations of lansoprazole.     

Within-day reproducibility of an HPLC-MS-based method for metabonomic analysis: application to human urine

Self-evidently, research in areas supporting "systems biology" such as genomics, proteomics, and metabonomics are critically dependent on the generation of sound analytical data. Metabolic phenotyping using LC-MS-based methods is currently at a relatively early stage of development, and approaches to ensure data quality are still developing. As part of studies on the application of LC-MS in metabonomics, the within-day reproducibility of LC-MS, with both positive and negative electrospray ionization (ESI), has been investigated using a standard "quality control" (QC) sample. The results showed that the first few injections on the system were not representative, and should be discarded, and that reproducibility was critically dependent on signal intensity. On the basis of these findings, an analytical protocol for the metabonomic analysis of human urine has been developed with proposed acceptance criteria based on a step-by-step assessment of the data. Short-term sample stability for human urine was also assessed. Samples were stable for at least 20 h at 4 degrees C in the autosampler while queuing for analysis. Samples stored at either -20 or -80 degrees C for up to 1 month were indistinguishable on subsequent LC-MS analysis. Overall, by careful monitoring of the QC data, it is possible to demonstrate that the "within-day" reproducibility of LC-MS is sufficient to ensure data quality in global metabolic profiling applications.     

An approach towards method development for untargeted urinary metabolite profiling in metabonomic research using UPLC/QToF MS

The application of LC-MS for untargeted urinary metabolite profiling in metabonomic research has gained much interest in recent years. However, the effects of varying sample pre-treatments and LC conditions on generic metabolite profiling have not been studied. We aimed to evaluate the effects of varying experimental conditions on data acquisition in untargeted urinary metabolite profiling using UPLC/QToF MS. In-house QC sample clustering was used to monitor the performance of the analytical platform. In terms of sample pre-treatment, results showed that untreated filtered urine yielded the highest number of features but dilution with methanol provided a more homogenous urinary metabolic profile with less variation in number of features and feature intensities. An increased cycle time with a lower flow rate (400mul/min vs 600mul/min) also resulted in a higher number of features with less variability. The step elution gradient yielded the highest number of features and the best chromatographic resolution among three different elution gradients tested. The maximum retention time and mass shift were only 0.03min and 0.0015Da respectively over 600 injections. The analytical platform also showed excellent robustness as evident by tight QC sample clustering. To conclude, we have investigated LC conditions by studying variability and repeatability of LC-MS data for untargeted urinary metabolite profiling.     

Evaluating the influence of quality control decisions and software algorithms on SNP calling for the affymetrix 6.0 SNP array platform

Objective: Our goal was to evaluate the influence of quality control (QC) decisions using two genotype calling algorithms, CRLMM and Birdseed, designed for the Affymetrix SNP Array 6.0. Methods: Various QC options were tried using the two algorithms and comparisons were made on subject and call rate and on association results using two data sets. Results: For Birdseed, we recommend using the contrast QC instead of QC call rate for sample QC. For CRLMM, we recommend using the signal-to-noise rate ≥4 for sample QC and a posterior probability of 90% for genotype accuracy. For both algorithms, we recommend calling the genotype separately for each plate, and dropping SNPs with a lower call rate (<95%) before evaluating samples with lower call rates. To investigate whether the genotype calls from the two algorithms impacted the genome-wide association results, we performed association analysis using data from the GENOA cohort; we observed that the number of significant SNPs were similar using either CRLMM or Birdseed. Conclusions: Using our suggested workflow both algorithms performed similarly; however, fewer samples were removed and CRLMM took half the time to run our 854 study samples (4.2 h) compared to Birdseed (8.4 h).     

Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies

Background

Quality assurance (QA) and quality control (QC) are two quality management processes that are integral to the success of metabolomics including their application for the acquisition of high quality data in any high-throughput analytical chemistry laboratory. QA defines all the planned and systematic activities implemented before samples are collected, to provide confidence that a subsequent analytical process will fulfil predetermined requirements for quality. QC can be defined as the operational techniques and activities used to measure and report these quality requirements after data acquisition.

Aim of review

This tutorial review will guide the reader through the use of system suitability and QC samples, why these samples should be applied and how the quality of data can be reported.

Key scientific concepts of review

System suitability samples are applied to assess the operation and lack of contamination of the analytical platform prior to sample analysis. Isotopically-labelled internal standards are applied to assess system stability for each sample analysed. Pooled QC samples are applied to condition the analytical platform, perform intra-study reproducibility measurements (QC) and to correct mathematically for systematic errors. Standard reference materials and long-term reference QC samples are applied for inter-study and inter-laboratory assessment of data.

     

Cross validation and ruggedness testing of analytical methods used for the quantification of urinary phthalate metabolites

Since the publication of our first analytical method in 2000 to detect and quantify phthalate metabolites in human urine, we have modified the method several times to improve performance, reduce the volume of matrix and solvents used, and to increase the number of analytes in one analytical run. We performed cross method validation and ruggedness testing after each modification to ensure that the analytical method adopted is robust and produces accurate and reproducible data when compared to the previously used method. Here, we present the results from the evaluation of the ruggedness of our analytical approach under variable experimental conditions, using the current analytical method. Minor deviations of the standard experimental conditions, i.e., pH, incubation time, amount of deconjugation enzyme, and incubation temperature, had no effect on final analyte concentrations. Furthermore, we validated the method to ensure accuracy at concentrations beyond the highest calibration standard. The concentrations obtained by using a lower volume of urine agreed well with original levels, suggesting broad linear calibration range as well as complete hydrolysis of the glucuronide conjugates with the standard amount of beta-glucuronidase used for deglucuronidation; also, the time of incubation (90 min) was adequate regardless of the amount of glucuronide present. We also summarize the precision of concentration data acquired by the five different analytical approaches we have used since 2000. The correlation plots of concentration data for each analyte obtained from split sample analysis, using three of these approaches, produced linear curves (R(2)>0.98) with slopes and intercepts that were not statistically different (p>0.05) from 1 and 0, respectively. These results suggest that the data are reproducible and accurate, regardless of the analytical method used. Furthermore, analysis of quality control urine samples made over the years confirmed the stability of the phthalate metabolites in urine at -70 degrees C for several years and the consistency of the analytical measurements obtained by using various methodological approaches over time.     

An approach for the development and selection of chromatographic methods for high-throughput metabolomic screening of urine by ultra pressure LC-ESI-ToF-MS

Since the components of a sample for open metabolomic analysis are unknown a priori a pragmatic approach to method development has been taken in order to develop and select a chromatographic method suitable for high-throughput open metabolomic screening of urine by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS). A total of 848 injections of diluted rat urine were made onto a UPLC-ESI-ToF-MS system using several different gradient profiles and run times to determine a suitable method for analysis of urine from male and female rats. Peak integral and multivariate data analysis were performed to investigate the quality of separation and information obtained from these multiple analyses. A suitable 8 min method was selected and is now used routinely for open profiling metabolomic analyses of urine. The use of a sample-relevant QC mix is also discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development and validation of a liquid chromatography-mass spectrometry assay for the determination of pyronaridine in human urine

A reliable method has been developed for the determination of pyronaridine in human urine using amodiaquine as an internal standard. Liquid-liquid extraction was used for sample preparation. Analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. The extracted ion for pyronaridine was m/z 518.20 and for amodiaquine was m/z 356.10. Chromatography was carried out using a Gemini 5 microm C18 3.0 mmx150 mm column using 2 mM perflurooctanoic acid and acetonitrile mixture as a mobile phase delivered at a flow rate of 0.5 mL/min. The mobile phase was delivered in gradient mode. The retention times of pyronaridine and amodiaquine were 9.1 and 8.1 min respectively, with a total run time of 14 min. The assay was linear over a range of 14.3-1425 ng/mL for pyronaridine (R2>or=0.992, weighted 1/Concentration). The analysis of quality control samples for pyronaridine at 28.5, 285, 684 and 1140 ng/mL demonstrated excellent precision with relative standard deviation of 5.1, 2.3, 3.9 and 9.2%, respectively (n=5). Recoveries at concentrations of 28.5, 285, 684 and 1140 ng/mL were all greater than 85%.This LC-MS method for the determination of pyronaridine in human urine has excellent specifications for sensitivity, reproducibility and accuracy and can reliably quantitate concentrations of pyronaridine in urine as low as 14.3 ng/mL. The method will be used to quantify pyronaridine in human urine for pharmacokinetic and drug safety studies.     

High-throughput profiling of dietary polyphenols and their metabolites by HPLC-ESI-MS-MS in human urine

Polyphenols in biological fluids are generally estimated by HPLC with UV, electrochemical or fluorimetric detection. We describe herein a method specially developed to estimate biomarkers of polyphenol consumption in human urine. We simultaneously quantified 15 polyphenols and their related compounds in human urine using an HPLC coupled with electrospray ionization mass-mass (HPLC-ESI-MS-MS) method with an analytical run time of 6 min. The method has been validated with respect to linearity, precision, and accuracy in intra- and inter-day assays. It has been applied to human urine samples collected from volunteers after consumption of different polyphenol-rich beverages in controlled conditions.     

Determination of azatadine in human plasma by liquid chromatography/tandem mass spectrometry

A sensitive method using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) was developed and validated for the analysis of antihistamine drug azatadine in human plasma. Loratadine was used as internal standard (IS). Analytes were extracted from human plasma by liquid/liquid extraction using ethyl acetate. The organic phase was reduced to dryness under a stream of nitrogen at 30 °C and the residue was reconstituted with the mobile phase. 5 μL of the resulting solution was injected onto the LC-MS/MS system. A 4.6 mm × 150 mm, I.D. 5 μm, Agilent TC-C(18) column was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 0.010 M (adjusted to pH 4.3 with 1M formic acid)/acetonitrile (20:80, v/v) The chromatographic run time was 5 min per injection and flow rate was 0.6 mL/min. The retention time was 2.4 and 4.4 min for azatadine and IS, respectively. The tandem mass spectrometric detection mode was achieved with electrospray ionization (ESI) iron source and the multiple reaction monitoring (MRM) (291.3 → 248.2m/z for azatadine, 383.3 → 337.3m/z for IS) was operated in positive ion modes. The low limit of quantitation (LLOQ) was 0.05 ng/mL. The intra-day and inter-day precision of the quality control (QC) samples was 8.93-11.57% relative standard deviation (RSD). The inter-day accuracy of the QC samples was 96.83-105.07% of the nominal values.     

Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry

For the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous determination of ribavirin and rabavirin base was developed and validated over the concentration range of 10-5,000 ng/ml, respectively, using a 0.025 ml monkey plasma sample. Ribavirin, ribavirin base, and the internal standards were extracted from monkey plasma via protein precipitation. After evaporation of the supernatant, the extract was reconstituted with 5% methanol (containing 0.1% formic acid) and injected onto the LC-MS/MS system. Optimum chromatographic separation was achieved on a Waters Atlantis dc18 (150 mm x 2.1mm, 5 microm) column with mobile phase run in gradient with 100% water containing 0.5% formic acid (A) and 90% acetonitrile (containing 0.5% formic acid (B). The flow rate was 0.4-0.6 ml/min with total cycle time of approximately 7.0 min. Post-column addition of acetonitrile (containing 0.1% formic acid) at 0.3 ml/min was used to increase the ionization efficiency in the MS source. The method was validated for sensitivity, linearity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The intra-day and inter-day precision and accuracy of the quality control (QC) samples were <9.0% relative standard deviation (R.S.D.) and 10.8% bias for ribavirin, and 10.3% R.S.D. and 11.3% bias for ribavirin base. The current specific, accurate and precise assay is useful in support of the toxicokinetic and pharmacokinetic studies of these compounds.     

Analysis of goldenseal, Hydrastis canadensis L., and related alkaloids in urine using HPLC with UV detection

A screening method was developed to extract and detect berberine and hydrastine alkaloids from goldenseal root powder and urine samples using HPLC with UV detection. The isocratic method was developed to detect alkaloids in 5 mL of urine prior to drug screening. Urine samples were spiked with the alkaloids at varying concentrations and extracted twice with 3:1 chloroform:2-propanol (CHCl(3):2-propanol). The extracts were combined, concentrated using nitrogen gas and the residue was then reconstituted with a mobile phase of acetonitrile:buffer (32:68). A 17 min isocratic run time was performed with a flow rate of 2.0 mL/min, and UV detection at 230 nm using a C(18) (250 mm × 4.6 mm) column at room temperature. The method showed good linearity for berberine (r(2)=0.9990) and hydrastine (r(2)=0.9983) over a range of 11.80 ng/mL to 17.64 μg/mL. The LOD for berberine in urine was 12.74 ng/mL and the LOD for hydrastine in urine was 54.48 ng/mL. Urine samples were spiked with goldenseal root powder and liquid extract as part of a blinded study to determine whether berberine and hydrastine alkaloids could also be extracted in vitro from goldenseal and show a presence in urine samples. Out of the 37 blinded urine samples extracted the two spiked samples were correctly identified based on the presence or absence of berberine and hydrastine. The results demonstrated that this method will enable laboratories to test for the herbal supplement in submitted urine samples prior to drug testing, avoiding false negative results.     

Determination of triamterene and its main metabolite hydroxytriamterene sulfate in human urine by capillary electrophoresis using ultraviolet absorbance and laser-induced fluorescence detection

Two capillary electrophoresis methods have been developed for the direct determination of triamterene and its main metabolite hydroxytriamterene sulfate in human urine. Analytes were detected using conventional UV detection as well as laser-induced fluorescence (LIF) detection with an HeCd-laser operating at a wavelength of 325 nm. The results of both detection techniques were compared. Indeed, the limit of quantification was eightfold lower using LIF detection (50 ng/ml) in comparison to UV detection (400 ng/ml). As no interference due to endogenous urine compounds was observed, direct urine analysis was feasible. Analysis was very simple and fast-one run could be performed within less than 10 min (CE-UV method) and 2.5 min (CE-LIF method), respectively. Both assays were fully validated and applied to urine samples from a human volunteer. The results of the application of the CE-LIF method to human urine samples are presented in this publication.     

Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction

We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.     

Quantitative determination of BAF312, a S1P-R modulator,in human urine by LC–MS/MS: Prevention and recovery of lost analyte due to container surface adsorption

Analyte loss due to non-specific binding, especially container surface adsorption, is not uncommon in the quantitative analysis of urine samples. In developing a sensitive LC–MS/MS method for the determination of a drug candidate, BAF312, in human urine, a simple procedure was outlined for identification, confirmation and prevention of analyte non-specific binding to a container surface and to recover the ‘non-specific loss’ of an analyte, if no transfer has occurred to the original urine samples. Non-specific binding or container surface adsorption can be quickly identified by using freshly spiked urine calibration standards and pre-pooled QC samples during a LC–MS/MS feasibility run. The resulting low recovery of an analyte in urine samples can be prevented through the use of additives, such as the non-ionic surfactant Tween-80, CHAPS and others, to the container prior to urine sample collection. If the urine samples have not been transferred from the bulk container, the ‘non-specific binding’ of an analyte to the container surface can be reversed by the addition of a specified amount of CHAPS, Tween-80 or bovine serum albumin, followed by appropriate mixing. Among the above agents, Tween-80 is the most cost-effective. β-cyclodextrin may be suitable in stabilizing the analyte of interest in urine via pre-treating the matrix with the agent. However, post-addition of β-cyclodextrin to untreated urine samples does not recover the ‘lost’ analyte due to non-specific binding or container surface adsorption. In the case of BAF312, a dynamic range of 0.0200–20.0 ng/ml in human urine was validated with an overall accuracy and precision for QC sample results ranging from ?3.2 to 5.1% (bias) and 3.9 to 10.2% (CV), respectively. Pre- and post-addition of 0.5% (v/v) Tween-80 to the container provided excellent overall analyte recovery and minimal MS signal suppression when a liquid–liquid extraction in combination with an isocratic LC separation was employed. The compound was stable in 0.5% Tween-80 treated human urine QC samples for at least 24 h at room temperature, after three freeze/thaw cycles with storage at ≤?60 °C and for at least 3 months when stored at ≤?60 °C. The current work could serve as a simple example in trouble shooting non-specific binding or container surface adsorption in quantitative analysis of urine samples.     

Protein analysis by isoelectric focusing in a capillary array with an absorption imaging detector

Isoelectric focusing (IEF) was successuflly performed in capillary arrays with up to four capillaries. Separated proteins in the capillary array were detected by an UV absorption imaging detector. The whole analysis time for all samples in the capillary array was only 3 min due to the real-time imaging detector. The instrument was applied to analyse several protein samples including different human hemoglobin variants, myoglobin, transferrin, carbonic anhydrase and a monoclonal antibody to fluorescein. Because of good reproducibility of the focused pattern, unknown samples can be run simultaneously with a standard in the multichannel instrument and the components of unknown samples can be identified by comparing their zone positions to those of the standard. Minor components can be determined by the instrument in the presence of major components with 100 times higher concentrations in human hemoglobin samples. This instrument could be a powerful analytical tool for clinical analysis and for quality control in pharmaceutical companies.     

Quantification of total and free mycophenolic acid in human plasma by liquid chromatography with fluorescence detection

A simple high-performance liquid chromatographic (HPLC) method was developed for the assay of total and free mycophenolic acid (MPA) in human plasma. Prior to analysis, total mycophenolic acid was extracted by protein precipitation and free drug was isolated from plasma samples using ultrafiltration. The extracts were injected onto a Kromasil C8 column at 30 degrees C with excitation and emission wavelengths set at 342 and 425 nm, respectively. The mobile phase was consisted of acetonitrile-32 mM glycine buffer, pH 9.2 (20:80, v/v), at a flow rate of 1.0 ml/min. The method was found to be linear over the concentration range investigated, 0.05-40 mg/l for total mycophenolic acid (r>0.999) and 5-1000 microg/l (r>0.99) for free drug. The percentage error of the analytical method was below 10.9%. The intra- and inter-day reproducibility was adequate with the coefficients of variation of 8.28% or below. The run time were 4 and 6 min for free and total MPA, respectively. The method thus can be effectively applied to measure mycophenolic acid concentrations in clinical samples.     

Metabolic profiling of serum using Ultra Performance Liquid Chromatography and the LTQ-Orbitrap mass spectrometry system

Advances in analytical instrumentation can provide significant advantages to the volume and quality of biological knowledge acquired in metabolomic investigations. The interfacing of sub-2mum liquid chromatography (UPLC ACQUITY((R))) and LTQ-Orbitrap mass spectrometry systems provides many theoretical advantages. The applicability of the interfaced systems was investigated using a simple 11-component metabolite mix and a complex mammalian biofluid, serum. Metabolites were detected in the metabolite mix with signals that were linear with their concentration over 2.5-3.5 orders of magnitude, with correlation coefficients greater than 0.993 and limits of detection less than 1mumolL(-1). Reproducibility of retention time (RSD<3%) and chromatographic peak area (RSD<15%) and a high mass accuracy (<2ppm) were observed for 14 QC serum samples interdispersed with other serum samples, analysed over a period of 40h. The evaluation of a single deconvolution software package (XCMS) was performed and showed that two parameters (snthresh and bw) provided significant changes to the number of peaks detected and the peak area reproducibility for the dataset used. The data were used to indicate possible biomarkers of pre-eclampsia and showed both the instruments and XCMS to be applicable to the reproducible and valid detection of disease biomarkers present in serum.