Jack Henion

Liquid extraction surface analysis mass spectrometry (LESA-MS) as a novel profiling tool for drug distribution and metabolism analysis: the terfenadine example.

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a novel surface profiling technique that combines micro-liquid extraction from a solid surface with nano-electrospray mass spectrometry. One potential application is the examination of the distribution of drugs and their metabolites by analyzing ex vivo tissue sections, an area where quantitative whole body autoradiography (QWBA) is traditionally employed. However, QWBA relies on the use of radiolabeled drugs and is limited to total radioactivity measured whereas LESA-MS can provide drug- and metabolite-specific distribution information. Here, we evaluate LESA-MS, examining the distribution and biotransformation of unlabeled terfenadine in mice and compare our findings to QWBA, whole tissue LC/MS/MS and MALDI-MSI. The spatial resolution of LESA-MS can be optimized to ca. 1 mm on tissues such as brain, liver and kidney, also enabling drug profiling within a single organ. LESA-MS can readily identify the biotransformation of terfenadine to its major, active metabolite fexofenadine. Relative quantification can confirm the rapid absorption of terfendine after oral dosage, its extensive first pass metabolism and the distribution of both compounds into systemic tissues such as muscle, spleen and kidney. The elimination appears to be consistent with biliary excretion and only trace levels of fexofenadine could be confirmed in brain. We found LESA-MS to be more informative in terms of drug distribution than a comparable MALDI-MS imaging study, likely due to its favorable overall sensitivity due to the larger surface area sampled. LESA-MS appears to be a useful new profiling tool for examining the distribution of drugs and their metabolites in tissue sections.

Microsample Determination of Lovastatin and its Hydroxy Acid Metabolite in Mouse and Rat Plasma by Liquid Chromatography/Ionspray Tandem Mass Spectrometry

A sensitive and specific method was developed and validated to quantitate lovastatin and its hydroxy acid in mouse and rat plasma. This method employs a solid-phase extraction procedure to isolate lovastatin and its hydroxy acid metabolite from the biological matrices (0.1 ml of mouse or rat plasma). The reconstituted extracts were analyzed by liquid chromatography/ionspray tandem mass spectrometry (LC/MS/MS). Simvastatin and simvastatin hydroxy acid were used as internal standards for lovastatin and lovastatin hydroxy acid, respectively. The assay has a lower limit of quantitation (LLQ) of 0.50 ng ml-1 in mouse and rat plasma for both lovastatin and its hydroxy acid based on 0.1 ml aliquots of plasma. The intra- and inter-assay precision (RSD), calculated from quality control (QC) samples, was < 7% for lovastatin and < 6% for lovastatin hydroxy acid in both matrices. The inter-assay accuracy as determined from QC samples was less than 6% for lovastatin and less than 8% for lovastatin hydroxy acid in both matrices. The overall recovery of lovastatin was 54% in mouse plasma and 55% in rat plasma, and the overall recovery of lovastatin hydroxy acid was 100% in mouse plasma and 67% in rat plasma.

The use of a membrane filtration device to form dried plasma spots for the quantitative determination of guanfacine in whole blood.

RATIONALEnA two-layered polymeric membrane is employed for the formation of separated dried plasma spots from whole blood as an alternative to the direct analysis of whole dried blood spots (DBS). This dried plasma spot (DPS) analysis procedure precludes potential issues of hematocrit differences in whole blood samples while providing pharmokinetic data from plasma rather than whole blood. The described procedure is also semi-automated thus providing a simpler work flow for LC/MS/MS bioanalysis procedures.nnnMETHODSnMolecular filtration of red blood cells (RBC) from applied microsamples of whole blood fortified with guanfacine and its stable isotope internal standard was accomplished with a two-layer polymeric membrane substrate. The lower membrane surface containing the separated plasma spot was physically separated from the upper membrane followed by semi-automated direct elution of the sample to an online solid-phase extraction (SPE) cartridge followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS).nnnRESULTSnA two-layer membrane sample preparation substrate produced plasma from whole blood without centrifugation which could be directly eluted for semi-automated LC/MS/MS bioanalysis. Standard curves were constructed by plotting peak area ratios between the analyte and the stable isotope labeled internal standard (SIL-IS) versus the nominal concentration in whole blood. A weighted 1/x(2) linear regression was applied to the data from DPS samples. Standard curves were linear over the range 0.25-250 ng/mL human whole blood. The representative regression equation was y = 0.0142x + 0.00248 (R(2) = 0.995) for the described DPS assay.nnnCONCLUSIONSnThe described work demonstrates proof-of-principle using membrane sample preparation techniques to form DPS samples from whole blood for subsequent bioanalysis by LC/MS/MS. This approach has the potential to eliminate the hematocrit issues from the current controversy surrounding validation of DBS assays.

Liquid extraction surface analysis (LESA) of food surfaces employing chip‐based nano‐electrospray mass spectrometry

An automated surface-sampling technique called liquid extraction surface analysis (LESA), coupled with infusion nano-electrospray high-resolution mass spectrometry and tandem mass spectrometry (MS/MS), is described and applied to the qualitative determination of surface chemical residues resulting from the artificial spraying of selected fresh fruits and vegetables with representative pesticides. Each of the targeted pesticides was readily detected with both high-resolution and full-scan collision-induced dissociation (CID) mass spectra. In the case of simazine and sevin, a mass resolution of 100,000 was insufficient to distinguish the isobaric protonated molecules for these compounds. When the surface of a spinach leaf was analyzed by LESA, trace levels of diazinon were readily detected on the spinach purchased directly from a supermarket before they were sprayed with the five-pesticide mixture. A 30 s rinse under hot running tap water appeared to quantitatively remove all remaining residues of this pesticide. Diazinon was readily detected by LESA analysis on the skin of the artificially sprayed spinach. Finally, incurred pyrimethanil at a level of 169 ppb in a batch slurry of homogenized apples was analyzed by LESA and this pesticide was readily detected by both high-resolution mass spectrometry and full-scan CID mass spectrometry, thus showing that pesticides may also be detected in whole fruit homogenized samples. This report shows that representative pesticides on fruit and vegetable surfaces present at levels 20-fold below generally allowed EPA tolerance levels are readily detected and confirmed by the title technologies making LESA-MS as interesting screening method for food safety purposes.

Quantitative liquid chromatographic-tandem mass spectrometric determination of reserpine in FVB/N mouse plasma using a chelating agent (disodium EDTA) for releasing protein-bound analytes during 96-well liquid-liquid extraction

A sensitive, specific, accurate and reproducible analytical method employing a divalent cation chelating agent (disodium EDTA) for sample treatment was developed to quantitate reserpine in FVB/N mouse plasma. Samples pretreated with 40 microl of 2% disodium EDTA in water were extracted by a semi-automated 96-well liquid-liquid extraction (LLE) procedure to isolate reserpine and a structural analog internal standard (I.S.), rescinnamine, from mouse plasma. The extracts were analyzed by turbo ionspray liquid chromatography-tandem mass spectrometry (LC-MS-MS) in the positive ion mode. Sample preparation time for conventional LLE was dramatically reduced by the semi-automated 96-well LLE approach. The assay demonstrated a lower limit of quantitation of 0.02 ng/ml using 0.1-ml plasma sample aliquots. The calibration curves were linear from 0.02 to 10 ng/ml for reserpine. The intra- and inter-assay precision of quality control (QC) samples ranged from 1.75 to 10.9% for reserpine. The intra- and inter-assay accuracy of QC samples ranged from -8.17 to 8.61%. Reserpine and the I.S. were found to be highly bound to FVB/N mouse plasma protein. This is the first report of disodium EDTA employed as a special protein-bound release agent to recover protein-bound analytes from plasma. These matrix effects and the effects of pH in the HPLC mobile phase on the sensitivities of LC-MS-MS are discussed in this paper.

Quantitative Determination of Orlistat(Tetrahydrolipostatin, Ro 18-0647) in HumanPlasma by High-performance LiquidChromatography Coupled with Ion Spray Tandem Mass Spectrometry

A rapid, sensitive and specific analytical method was developed and validated to quantify tetrahydrolipostatin (Orlistat, Ro 18-0647) in human plasma in order to provide pharmacokinetic data from clinical trials. This method employs a preliminary plasma protein precipitation step followed by a simple, one-step liquid-liquid extraction procedure to isolate Ro 18-0647 and its pentadeuterated internal standard, Ro 18-0647-d5, from the biological matrix. Reconstituted extracts were analyzed by liquid chromatography/ion spray tandem mass spectrometry (LC/MS/MS) in the selected reaction monitoring (SRM) mode. Chromatography was carried out using a 2 mm i.d. x 50 mm Deltabond Phenyl column. The eluent was acetonitrile-2 mM ammonium acetate (90:10). The retention time of the analyte was 1.2 min and chromatographic run times were less than 1.5 min. No interferences from anticoagulants, collection devices or endogenous constituents of the plasma were observed. The assay has a lower limit of quantitation (LLQ) of 0.20 ng ml-1 in plasma and a lower limit of detection (LLD) of 0.10 ng ml-1 plasma, based on 1 ml aliquots. The capability to detect 0.025 ng ml-1 in plasma has also been demonstrated. The calibration graphs were linear from 0.20 to 10 ng ml-1. The assay was initially validated with a linear range of 0.20-1.0 ng ml-1. This range was later extended and validated to an upper level of quantitation of 10 ng ml-1. Intra- and inter-assay precision studies showed a mean variability of less than 10%. The recovery, inter-assay precision and accuracy of the method were within acceptable bioanalytical standards. The assay has been shown to reliably provide automated, unattended sample analysis for approximately 150 samples per day. In an additional series of tests, Ro 18-0467 was shown to be stable under conditions that might be encountered during the analysis of samples from clinical trials. This LC/MS/MS assay procedure for Ro 18-0647 in human plasma has proven to be robust, sensitive, specific, accurate and reproducible. This method has been used to analyze over 5000 study samples.

Microsample analyses via DBS: challenges and opportunities

The use of DBS is an appealing approach to employing microsampling techniques for the bioanalysis of samples, as has been demonstrated for the past 50 years in the metabolic screening of metabolites and diseases. In addition to its minimally invasive sample collection procedures and its economical merits, DBS microsampling benefits from the very high sensitivity, selectivity and multianalyte capabilities of LC-MS, which has been especially well demonstrated in newborn screening applications. Only a few microliters of a biological fluid are required for analysis, which also translates to significantly reduced demands on clinical samples from patients or from animals. Recently, the pharmaceutical industry and other arenas have begun to explore the utility and practicality of DBS microsampling. This review discusses the basis for why DBS techniques are likely to be part of the future, as well as offering insights into where these benefits may be realized.

Quantitative determination of opioids in whole blood using fully automated dried blood spot desorption coupled to on-line SPE-LC-MS/MS.

Opioids are well known, widely used painkillers. Increased stability of opioids in the dried blood spot (DBS) matrix compared to blood/plasma has been described. Other benefits provided by DBS techniques include point-of-care collection, less invasive micro sampling, more economical shipment, and convenient storage. Current methodology for analysis of micro whole blood samples for opioids is limited to the classical DBS workflow, including tedious manual punching of the DBS cards followed by extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. The goal of this study was to develop and validate a fully automated on-line sample preparation procedure for the analysis of DBS micro samples relevant to the detection of opioids in finger prick blood. To this end, automated flow-through elution of DBS cards was followed by on-line solid-phase extraction (SPE) and analysis by LC-MS/MS. Selective, sensitive, accurate, and reproducible quantitation of five representative opioids in human blood at sub-therapeutic, therapeutic, and toxic levels was achieved. The range of reliable response (R(2) u2009≥0.997) was 1 to 500u2009ng/mL whole blood for morphine, codeine, oxycodone, hydrocodone; and 0.1 to 50u2009ng/mL for fentanyl. Inter-day, intra-day, and matrix inter-lot accuracy and precision was less than 15% (even at lower limits of quantitation (LLOQ) level). The method was successfully used to measure hydrocodone and its major metabolite norhydrocodone in incurred human samples. Our data support the enormous potential of DBS sampling and automated analysis for monitoring opioids as well as other pharmaceuticals in both anti-doping and pain management regimens.

Dried blood spots as a sampling technique for the quantitative determination of guanfacine in clinical studies.

BACKGROUNDnDried blood spot (DBS) technology was evaluated for the quantitative determination of guanfacine in human blood in clinical studies. A very sensitive DBS assay has been developed using HPLC coupled with an AB Sciex 5500 QTRAP® (Applied Biosystems/MDS Sciex, ON, Canada) MS system (LC-MS/MS) with a linear calibration range of 0.05 to 25 ng/ml. High-resolution MS using an Exactive Orbitrap® (ThermoFisher, LLC., CA, USA) was compared with the QTRAP using extracted exact mass ion current profiles for guanfacine and its stable-isotope-incorporated internal standard. The sample preparation employed liquid-liquid extraction with methyl t-butyl ether of 5 mm punched DBS card disks, followed by reversed-phase HPLC separation coupled with either MS/MS or high-resolution MS. Routine experiments were performed to establish the robustness of the DBS assay, including precision, accuracy, linearity, selectivity, sensitivity and long-term stability of up to 76 days. In addition, several factors that potentially affect quantitation were investigated, including blood volume for DBS spotting, punch size and punch location.nnnRESULTSnA sensitive research assay with a LLOQ of 0.05 ng/ml was developed and subjected to several components of a method validation common to a regulated bioanalysis procedure employing DBS. This method development and partial validation study determined that spot volume, punch size or punch location do not affect assay accuracy and precision. The DBS approach was successfully applied to a clinical study (a Phase I, randomized, double-blind, placebo-controlled, crossover study to assess the effect of varying multiple oral doses of guanfacine on the pharmacokinetic, pharmacodynamic, safety, and tolerability profiles in healthy adult subjects). The pharmacokinetic profiles for 12 volunteers generated from the DBS assay and from a previously validated plasma assay were compared and were found to be comparable. DBS incurred samples collected from finger prick blood and directly applied to the DBS cards were also analyzed for comparison.nnnCONCLUSIONnFrom a bioanalytical perspective, DBS sample collection and analysis is a potentially viable alternative for guanfacine determination in clinical studies, utilizing approximately 100 µl of blood per subject profile compared with a few millilitres of blood drawn for conventional plasma bioanalysis.

Semi-automated direct elution of dried blood spots for the quantitative determination of guanfacine in human blood

BACKGROUNDnDirect analysis of dried blood spot (DBS) samples was investigated using a prototype semi-automated robotic device that allows the direct elution of sample spots from a DBS paper card to an online SPE cartridge. The eluted SPE samples were analyzed with high-performance LC-MS/MS.nnnRESULTSnA LLOQ of 0.01 ng/ml was achieved with a linear calibration range from 0.01 to 25 ng/ml. Optimal performance data were obtained from spotting the internal standard solution on the card before blood spotting. Internal standard addition from the system injector loop produced intra-assay inaccuracy of -9.0-7.3% and precision of 1.3-8.2%, and inter-assay inaccuracy of -3.5-3.9% and precision of 4.4-8.7%.nnnCONCLUSIONnResults demonstrated the feasibility of a semi-automated online rapid direct elution method that avoids manual extraction for DBS sample analysis using the online DBS-SPE system coupled to LC-MS/MS.