Use of generic fast gradient liquid chromatography-tandem mass spectroscopy in quantitative bioanalysis.

Short narrow analytical HPLC columns have been used successfully with high linear flow-rates and combined with mass spectrometric detection to produce a generic approach to quantitative bioanalysis. The approach has been used to validate several assays in the low ng/ml region and an example is given in this paper. When combined with a simple solid-phase extraction process the need for complicated, time consuming method development has been removed for the majority of pharmaceutical compounds. The approach takes advantage of not only the extra selectivity of the MS-MS detector but the excellent resolution and peak shape produced by gradient elution.

Application of a generic fast gradient liquid chromatography tandem mass spectrometry method for the analysis of cytochrome P450 probe substrates.

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the fast routine analysis of selected CYP450 probe substrate metabolites in microsomal incubations, with no sample pretreatment. This has allowed fast and simple assessment of the potential effects which drug candidates may or may not have on the metabolism of specific CYP450 probe substrates, providing information which can then be used to rationalize in vivo interaction studies required in the clinic. This methodology takes advantage of fast gradient chromatography as a generic means of sample separation and analysis. It provides high throughput analysis compared to conventional gradient HPLC, with no significant loss in chromatographic performance.

Optimisation and routine use of generic ultra-high flow-rate liquid chromatography with mass spectrometric detection for the direct on-line analysis of pharmaceuticals in plasma.

The use of ultra-high flow-rate chromatography coupled to mass spectrometry offers great potential for the rapid, on-line analysis of pharmaceutical compounds in plasma as it permits high throughput direct analysis of plasma samples without any time-consuming sample preparation such as solid-phase extraction. The coupling of mass spectrometry with high-performance liquid chromatography often results in enhanced selectivity and sensitivity compared to, for example, ultraviolet absorbance detection. This can remove the need for complete resolution of the analyte from endogenous materials in the matrix. The use of large particle size stationary phases, and therefore, the ability to use large porosity column end frits, coupled with the added selectivity and sensitivity of the mass spectrometer allows an on-line analysis approach to be used for the direct analysis of pharmaceuticals in biological matrices with extremely high throughput. This paper presents an overview of the manner in which we have optimised this technique for the analysis of plasma samples, in terms of gradient profile, system configuration and optimal injection volume for maximum throughput and robustness. The nature of the mobile phase flow is also discussed.

The absolute bioavailability and metabolic disposition of the novel antimigraine compound zolmitriptan (311C90).

AIMS: Two open studies in healthy volunteers were conducted to determine the absolute bioavailability and metabolic disposition of zolmitriptan (311C90), a novel 5HT1D agonist for the acute treatment of migraine. METHODS: After an initial test i.v. infusion, bioavailability was assessed by comparison of AUC after an i.v. infusion (3.5 mg) and an oral tablet (10 mg), in six men and six women using a randomised, crossover design. Disposition was studied by administration of a 25 mg capsule, labelled with 100 microCi [14C]-zolmitriptan, to five men and one woman on a single occasion. RESULTS: Zolmitriptan was well tolerated by both i.v. and oral routes. Adverse events were mostly mild, consistent with earlier studies and characteristic of this class of drug. Reports were similar in nature and number after both oral and i.v. dosing. Mean +/- s.d. oral bioavailability was 0.49 +/- 0.24 (0.38 +/- 0.16 in men and 0.60 +/- 0.28 in women). After oral dosing, Cmax and AUC values in women were approximately double those in men. Relative to zolmitriptan concentrations, metabolite concentrations were higher after oral dosing than after i.v., and higher in men compared with women. Half-life was significantly longer after oral dosing (mean 22%, 95% CI 6-35%). Mean +/- s.d. values for CL, V2 and t1/2,z after i.v. dosing (all subjects) were 8.7 +/- 1.7 ml min-1 kg-1, 122 +/- 321 and 2.30 +/- 0.59 h respectively. Following administration of 25 mg [14C]-zolmitriptan, 91.5% of the dose was recovered in 7 days, 64.4 +/- 6.5% in urine and 27.1 +/- 6.0% in faeces. Less than 10% was recovered unchanged in urine, with 31.1 +/- 6.4% recovered as the inactive indole acetic acid metabolite. Most of the faecal material was unchanged zolmitriptan, representing unabsorbed drug. Plasma concentrations of [14C] were slightly higher than those of the summed concentrations of known analytes zolmitriptan, the active N-desmethyl metabolite (183C91), the inactive N-oxide (1652W92) and indole acetic acid (2161W92) metabolites, which accounted for 86% of total plasma radioactivity. No other significant metabolites were detected in plasma. Some minor additional metabolites were detected in urine, none of which contributed more than 5% of the dose. CONCLUSIONS: The data suggest that zolmitriptan undergoes first-pass metabolism and this is more extensive in men than in women. Zolmitriptan has suitable bioavailability for an acute oral migraine treatment and there are no significant unidentified metabolites in man.

The use of turbulent flow chromatography/mass spectrometry for the rapid, direct analysis of a novel pharmaceutical compound in plasma.

The use of turbulent flow chromatography coupled to mass spectrometry (turbulent flow LC/MS) shows great potential for the rapid, direct analysis of pharmaceutical compounds in plasma and serum. The use of turbulent flow LC/MS has removed the need for any time-consuming sample preparation such as solid phase extraction, and allowed a total sample analysis time of approximately 2.5 min to be achieved. The coupling of a mass spectrometer with HPLC often not only results in greater sensitivity, but also the added specificity of the mass spectrometer reduces the need for complete resolution of the analyte from endogenous material in the matrix. This allows an on-line analysis approach to be used for the analysis of pharmaceuticals in biological matrices. Turbulent flow chromatography is achieved by the use of high flow rates and large particle size stationary phases. When coupled with mass spectrometric detection, the technique allows the direct analysis of plasma or serum samples with very rapid chromatography and, therefore, extremely high throughout. This work demonstrates the suitability of this technique for the validated analysis of biological samples for a novel isoquinoline pharmaceutical and offers some ideas on the future continued development, optimization and application of turbulent flow liquid chromatography.

Analysis of 2-cyano-1-methyl-3-[4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl phenyl]guanidine in dog plasma by liquid-solid extraction and reversed-phase high-performance liquid chromatography.

A method for the determination of 2-cyano-1-methyl-3-[4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin- 3- yl)phenyl]guanidine (SK&F94836, 1) in plasma is presented. The method involves liquid-solid extraction of the drug by C18 cassettes, with subsequent elution by an AASP LC module for determination by HPLC with UV detection. The assay is rapid, precise, accurate, and specific. The between-day CV values over the concentration range 50-500 ng/mL are 4% or less; this rises to 8% at 25 ng/mL. The corresponding between-day bias is less than 1% over the same range of concentrations. The limit of quantification is 25 ng/mL and the assay can be used for measuring 1 in samples from preclinical studies following either oral or intravenous administration of the compound.

The analysis of SK&F 94120, a novel inotropic agent, and its four metabolites by isolation on C18 AASP cassettes followed by high-performance liquid chromatography.

A selective and specific assay for SK&F 94120 [5-(4-acetamidophenyl)pyrazin-2(1H)-one] and its four metabolites in plasma has been developed. The method incorporates a single liquid-solid extraction step using C18 Analytichem Automated Sample Processor (AASP) cassettes. This is followed by successive elutions of the solid phase with two mobile phases of increasing acetonitrile content, by an AASP liquid chromatography module. A mobile phase containing 10% acetonitrile elutes a glucuronide metabolite from the cartridge which is then chromatographed and quantified intact. A second mobile phase, containing 20% acetonitrile, is then used to elute the unchanged drug and the three other metabolites from the same cartridge. The assay shows good accuracy and precision (less than 10% for all analytes) and is able to determine SK&F 94120 and its metabolites in plasma at concentrations between 0.05 and 1.00 mg l(-1).

Analysis of 5-(4-acetamidophenyl)pyrazin-2(1H)-one (SK&F 94120) in plasma with an Analytichem automated sample processor liquid chromatography module.

A selective and specific assay for 5-(4-acetamidophenyl)pyrazin-2(1H)-one (SK&F 94120), a novel inotropic agent, has been developed. The method incorporates a liquid-solid extraction step with a C18 Analytichem automated sample processor (AASP) cassette, which consists of ten miniature extraction columns. The cassette is then loaded into the AASP auto injector, ready for automated liquid chromatography with UV detection. The AASP consists of a high-pressure sealing chamber which encapsulates each column. The high-performance liquid chromatographic mobile phase is directed through the chamber, and the analytes are eluted onto the analytical column for subsequent separation and measurement. The assay is sufficiently accurate and precise to determine SK&F 94120 at concentrations as low as 0.5 mg/l. The mean coefficient of variation for the concentration range 0.5-10.0 mg/l was 2% with a bias of +/- 1%. The assay has been used for pharmacokinetic and bioavailability studies in several species, including rat, dog, and cynomolgus monkey.