J.D. Gilbert

The simultaneous determination of mixtures of drug candidates by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry as an in vivo drug screening procedure.

Liquid chromatography, combined with tandem mass spectrometry (LC/MS/MS) has been rapidly embraced by the pharmaceutical industry as the definitive method for the determination of drug levels in biological fluids obtained from pharmacokinetic and toxicological studies. This technique has proved to be reliable, accurate and precise for the determination of drugs and related substances (e.g. metabolites and isotope-labeled compounds) in support of preclinical and clinical studies. Our group has recently expanded the use of quantitative LC/MS/MS into the area of discovering new substances as potential drug candidates. When used as an accurate mass detector, triple quadrupole instruments have the ability to simultaneously and specifically detect minute quantities of closely-related drug substances in the extracts of biological fluids. Analytical procedures have been developed and validated that simultaneously determine plasma concentrations of up to 12 drug candidates over a concentration range of 1-1000 ng mL-1 in single analytical occasions. This approach is used to support drug discovery by rapidly providing pharmacokinetic data to a wide range of compounds following either the administration of multiple compounds to single animals, or by increasing the speed and efficiency of analyzing samples following the administration of single compounds to multiple animals. Currently, we have screened over 400 compounds in two different target classes in a period of 24 weeks. A standard operating procedure defining the acceptability of quality control data obtained during such screening experiments is described.

A direct technique for the simultaneous determination of 10 drug candidates in plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry interfaced to a Prospekt solid-phase extraction system.

New drug candidates are being synthesized at an ever increasing rate and, until recently, the pharmacokinetics of only a few of these could be evaluated. Our laboratory is taking a novel approach to rapid multiple pharmacokinetic screening of potential drug candidates in which mixtures of new substances are co-administered to animals and analyzed simultaneously in plasma using liquid chromatography with tandem MS/MS detection in conjunction with a Prospekt automated on-line solid-phase extraction system. Plasma is sampled via an autosampler and extracted by the Prospekt with the eluent being introduced directly via a reverse phase HPLC column and a heated nebulizer interface to the mass spectrometer. Generic extraction and chromatographic conditions generally give good recoveries. The chromatographic run-times are less than 8 min. The accuracy and precision of these assays are carefully controlled with recoveries generally in the range 80-120% and coefficients of variation less than 20%. Lower quantifiable limits range from 2.5 to 5 ng ml-1. This approach considerably reduces the number of animals needed to screen drug candidates and its power is illustrated by determination of the pharmacokinetics of 10 substances after their simultaneous administration to dogs.

Quantitation of the 5HT1D agonists MK-462 and sumatriptan in plasma by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

The 5HT1D agonist sumatriptan is efficacious in the treatment of migraines. MK-462 is a drug of the same class which is under development in our laboratories. Bioanalytical methods of high efficiency, specificity and sensitivity were required to support the preclinical and clinical programs. These assays were based on HPLC with tandem MS-MS detection. MK-462 and sumatriptan were extracted using an automated solid-phase extraction technique on a C2 Varian Bond-Elut cartridge. The n-diethyl analogues of MK-462 and sumatriptan were used as internal standards. The analytes were chromatographed using reversed-phase (nitrile) columns coupled via a heated nebulizer interface to an atmospheric pressure chemical ionization source. The chromatographic run times were less than 7 min. Both methods were precise, accurate and selective down to plasma concentrations of 0.5 ng/ml. The assay for MK-462 was adapted to separately monitor the unlabeled and 14C-labeled species of the drug following intravenous administration of radiolabeled material to man.

Time-resolved fluoroimmunoassay for the determination of lisinopril and enalaprilat in human serum

A solid-phase immunoassay with detection based on time-resolved fluorescence (TR-FIA) has been developed for the determination of lisinopril and enalaprilat in human serum. The immunogen was prepared by coupling lisinopril to bovine serum albumin through a two-step reaction with difluorodinitrobenzene. An antiserum specific to both lisinopril and enalaprilat was used. The assay is based on the competitive immunoassay principle in which the drug competes with biotin-labeled drug for a limited quantity of primary antibody bound via sheep anti-rabbit globulin to the wells of microtitration strips. At the end of the first incubation, the unbound biotin-labeled drug is washed away. In the second step, europium-labeled streptavidin (specific to biotin) reacts with the biotin already bound to the solid-phase antibody. After a washing step, the addition of an enhancement solution dissociates the europium ions from the labeled streptavidin into solution. The fluorescence from each sample is inversely proportional to the concentration of the drug in the sample. The assay demonstrates good accuracy, reproducibility and specificity at serum concentrations down to 0.5 ng ml-1. However, the useful concentration range of TR-FIA is much narrower than that obtained by double antibody radioimmunoassay (RIA).

The Use of Stable Isotope Labeling and Liquid Chromatography/Tandem Mass Spectrometry Techniques to Study the Pharmacokinetics and Bioavailability of the Antimigraine Drug, MK‐0462 (Rizatriptan) in Dogs

MK-0462 (rizatriptan) is a 5HT1D agonist being developed for the treatment of migraine. The assay for this substance in plasma and urine is based on HPLC with tandem mass spectrometry (MS/MS) detection. The procedure has been modified to include the simultaneous determination of the [triazole-13C2, 15N3-] stable-isotope-labelled analogue for which the lower quantifiable limit was 0.1 ng mL-1. The assay has been applied to study the pharmacokinetics of MK-0462 after simultaneous oral and intravenous administration of the drug and its stable-isotope-labelled analogue to dogs. The experiment afforded an estimate of plasma clearance concomitant with a precise measurement of the drugs oral bioavailability. The increasing use of LC-MS/MS in quantitative experiments may renew interest in stable isotopes as tools for pharmaceutical research.

Determination of the β-adrenergic blocker timolol in plasma by liquid chromatography—atmospheric pressure chemical ionization mass spectrometry

Abstract A method based on LC—MS—MS has been developed for the determination of timolol in plasma using the (CD 3 ) 3 -labelled species as the internal standard. Timolol is isolated from plasma by a simple solid-phase extraction and converted to its oxazolidin-2-one prior to analysis on a 50 × 4.6 mm reversed-phase high-performance liquid chromatography column packed with SynChropak, C 18 , 5 μm. The column eluate is passed by means of a heated nebulizer interface into a corona discharge atmospheric pressure chemical ionization source where the analyte and its internal standard are detected using multiple reaction monitoring (MRM). The very high specificity of this technique permits chromatographic run times of less than 2 min. The method has a lower quantifiable limit of 0.5 ng ml −1 , with intra- and inter-day relative standard deviations less than 10%, and enables the determination of timolol in plasma after ocular administration to volunteers.

The development and cross-validation of methods based on radioimmunoassay and LC/MS-MS for the quantification of the Class III antiarrhythmic agent, MK-0499, in human plasma and urine

An analytical method based on radioimmunoassay (RIA) has been developed for the determination of the antiarrhythmic agent, MK-0499, in plasma and urine. Owing to the potency of the drug, the specificity of this assay in human plasma could not be adequately determined using conventional RIA procedures. A highly specific procedure, based on LC/MS-MS, was developed to cross-validate the RIA. The lower quantifiable limits of the RIA and LC/MS-MS-based methods were 0.05 and 0.013 ng ml-1, respectively. Cross-validation data, compared using paired students t-test regression analysis, showed excellent correlation between methods. The mass spectrometric assay was also used to simultaneously measure plasma concentrations of unlabeled and 14C-labeled MK-0499 following administration of the drug at high specific activity to volunteers.

A rapid and specific assay, based on liquid chromatography—atmospheric pressure chemical ionization mass spectrometry, for the determination of MK-434 (a 5α-reductase inhibitor) and its metabolites in plasma

MK-434 is a new 5 alpha-reductase inhibitor. A sensitive and specific assay based on combined liquid chromatography-mass spectrometry (LC-MS) has been developed for the determination of this compound in plasma. The analyte was isolated from plasma by solid-phase extraction on a C18 cartridge. A related substance, L-654,066, was used as the internal standard. Extracts were separated on a 5-cm C18-reversed-phase high performance liquid chromatography column interfaced via the heated nebulizer probe to a corona discharge chemical ionization source. The mass spectrometer was operated in the positive ion MS-MS mode. The method had sufficient sensitivity, precision, accuracy, and selectivity for the analysis of clinical samples containing MK-434 and its two principal metabolites at concentrations in the range 0.5-50 ng ml-1. The chromatographic run time was < 5 min.

Determination of MK-383, a non-peptide fibrinogen receptor antagonist, in human plasma and urine by radioimmunoassay

MK-383 is a novel, non-peptide fibrinogen receptor antagonist. A sensitive and specific radioimmunoassay has been developed for the determination of this drug candidate in plasma and urine. The immunogen was prepared by coupling to albumin via the N-hydroxysuccinimide ester from which the radioligand was also prepared by reaction with [I125]iodotyrosine. The method was specific and no immunoreactive material other than the parent drug was detectable in plasma and urine from dosed volunteers. This direct assay, using 5 microliters of plasma or 0.5 microliter of urine, is sensitive to 1 and 10 ng ml-1, respectively, without matrix interference and has sufficient sensitivity, specificity, accuracy, and precision for the analysis of clinical samples.

Use of LC-MS/MS to cross-validate a radioimmunoassay for the fibrinogen receptor antagonist, Aggrastat (tirofiban hydrochloride) in human plasma

A method based on LC-MS/MS was developed for the determination of the fibrinogen-receptor antagonist Aggrastat in human plasma. The drug is isolated from plasma by liquid extraction and converted into its N-trifluoroacetyl derivative prior to analysis by HPLC with atmospheric pressure negative chemical ionization MS/MS detection. A structural analog is used as the internal standard and the lower quantifiable limit of the assay is 0.4 ng ml-1 with a relative standard deviation of 7%. This assay was used to cross-validate the existing immunoassay by analysis of plasma from patients receiving the drug. The specificity of the immunoassay was thereby confirmed.