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The coupling of ultra-performance liquid chromatography, operating at elevated pressures, to a linear ion trap mass spectrometer provides a high-performance system suitable for drug metabolite characterisation. This system demonstrates improved chromatographic efficiency and sensitivity and at the same time provides diagnostic MSn data often critical for metabolite structural assignment. The linear ion trap was capable of dealing with the high chromatographic efficiencies and hence narrow peak widths associated with 1.7 microm particle-packed column separations. Polarity switching and data-dependent MSn data were generated with ease, and applied to the identification of metabolites found in human plasma., (Copyright 2006 John Wiley & Sons, Ltd.)

Monolithic columns have been successfully used with steep gradient and high flow rates for the direct analysis of a candidate pharmaceutical compound in human plasma. The monolithic columns showed excellent robustness with nearly 300 20-microL injections of plasma (diluted 1:1 with water) being made onto one column without significant deterioration in performance. The system gave excellent sensitivity with a limit of quantification of 5 ng/mL being achieved. Unlike previous methods of direct analysis the monolithic columns showed excellent resolution even after nearly 300 plasma injections. The column performance was measured before and after the analysis of the plasma samples., (Copyright 2001 John Wiley & Sons, Ltd.)

Capillary high-performance liquid chromatography (HPLC; 300 microm i.d.) coupled to tandem mass spectrometry has been used to determine the concentration of 4-hydroxytamoxifen in mouse plasma in the pg/mL range following the administration of Tamoxifen. A limit of quantification (LOQ) of 100 pg/mL was achieved using only 25 microL of plasma. The on-column sensitivity was determined to be 100 fg. The column performance was determined isocratically before and after the assay and showed only a 15% reduction in performance after 70 injections of plasma extract. No significant peak band broadening was observed due to the mass spectrometer interface using a standard TurboIonspray source., (Copyright 2001 John Wiley & Sons, Ltd.)

Fast gradient capillary high performance liquid chromatography (HPLC) coupled to a mass spectrometer has been successfully used for the analysis of pharmaceutical compounds from biological matrices, in the femtogram on column range. In the work reported in this paper, the use of capillary HPLC, on the 180-micron internal diameter scale, has shown a 30-fold improvement in detection limits when compared to conventional 2-mm scale chromatography. The use of fast gradient elution resulted in a generic methodology which gave excellent chromatographic reproducibility and column longevity. This technique has been used in conjunction with simple protein precipitation, with no deleterious effect on either the column life or the chromatographic performance. The use of capillary HPLC in bioanalysis has the potential to give a significant increase in assay sensitivity with the equipment currently in use. In this paper the authors also present a modification to the current PE-Sciex ion-spray source which allows excellent spray adjustment in three dimensional accessibility, which is important when working with low flow rates, as well as reducing the inherent system dead volume.

Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.

An automated mass spectrometric linked fraction collection system is described which enables on-line examination of isolated components based on molecular-ion and product-ion information. This system has been used successfully to specifically isolate drug-related material from complex biological fluids to aid structural identification. Metabolites have been isolated with sufficient purity to allow unequivocal characterisation by 1H nuclear magnetic resonance spectroscopy. This system has the advantage that isolation of the components of interest is not triggered by a simple contact closure. Therefore fraction collection is not biased by limitations in either the detector (e.g. insufficient sensitivity) or the analyst (e.g. programmed collection of predicted metabolites only). Furthermore, all isolated components are readily available post fractionation for additional screening.

Ultra-high flow rate liquid chromatography on large particle size stationary phases coupled with mass spectrometric detection (particularly tandem mass spectrometry, MS/MS) is gaining increasing usage for the direct determination of pharmaceuticals in biological fluids. The lack of sample preparation required prior to chromatographic and MS/MS analysis, together with the extremely high throughput of the chromatography, make the technique extremely attractive to the modern pharmaceutical bioanalyst. However, this lack of sample preparation also means that there is no potential for concentration of the sample and, as a consequence, the sensitivity of the technique has been limited. Liquid chromatography on the capillary scale offers sensitivity benefits compared with conventional liquid chromatography as the volume in which the analyte peaks are eluted is greatly reduced. In this paper, we present the use of ultra-high flow rate liquid chromatography on the capillary scale. This enables the quantification of drugs in plasma at sub-nanogram per millilitre concentrations from a very small (2.5 micromgL) aliquot of plasma without sample preparation. We also compare the resolution obtained by ultra-high flow rate liquid chromatography with that achieved on short columns packed with conventional size packing materials operated in an isocratic manner., (Copyright 1999 John Wiley & Sons, Ltd.)

The methodology described demonstrates, for the first time, the feasibility of performing pharmacokinetic studies in the serially bled mouse model to support the early development of discovery compounds. Sample analysis, using capillary high performance liquid chromatography combined with tandem mass spectrometry, has facilitated the achievement of this milestone and has successfully been applied to determine pharmacokinetic information following both intravenous and oral administration of a single discovery compound. The methodologies described demonstrate potential for a reduction in the amount of new chemical entity required to undertake pharmacokinetic studies. Typically, such studies are performed in larger rodents with a significantly increased body mass (ten times in the case of the rat) and therefore it follows that to undertake the same experiment in the mouse would require ten times less compound to effect an equivalent dose. Conventionally, pharmacokinetic studies to obtain both intravenous and oral information, e.g. clearance and half-life, and the resultant bioavailability have been performed using two parallel groups of rodents, collecting blood by exsanguination, separating off the plasma and analysing this using conventional liquid chromatography/tandem mass spectrometry. The use of capillary high performance liquid chromatography (HPLC) has facilitated the analysis of small volume blood samples by increasing the effective sensitivity of the analytical method. Consequently, we have established a protocol for serially bleeding mice thus reducing the number of animals and so further reducing the amount of compound required for such experiments. This paper reports data obtained from collected and processed blood volumes of <20 microL with the subsequent injection of only 1 microL of precipitated extract onto a capillary column., (Copyright 1999 John Wiley & Sons, Ltd.)

Preparative high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry has been used successfully for the isolation of several drug metabolites from urine. Nuclear magnetic resonance (NMR) spectroscopy has been employed to determine the exact chemical structure of these metabolites. The use of preparative HPLC has allowed the isolation of relatively large quantities of drug metabolites (> 0.5 mg) allowing insensitive, information-rich NMR experiments such as NOE, HMBC and HMQC to be performed. The coupling of the ion-trap mass spectrometer, operating in automatic MS/MS mode, to preparative HPLC allows the simultaneous collection and mass spectrometric analysis of eluting analytes to be performed, thus allowing the position of fractions containing drug-related material to be identified very rapidly.

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.

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.

Cisatracurium, (1R, 1'R, 2R, 2'R)-2,2-[1,5-pentanediylbis-[oxy(3-oxo- 3,1-propanediyl]]bis[1-[(3,4-dimethoxyphenyl)-methyl]-1,2,3,4- tetrahydro-6,7-dimethoxy-2-methylisoquinolinium] dibenzenesulphonate (51W89), is an intermediate-acting neuromuscular blocking agent. 51W89 is one of ten isomers contained in Tracrium (atracurium besylate) and represents approximately 15 percent of the atracurium mixture. Clinical studies have indicated that 51W89 is more potent and is significantly weaker as a histamine releaser than atracurium. In vitro studies in human plasma have shown that, like atracurium, 51W89 spontaneously degrades at physiological pH by Hoffmann elimination to form laudanosine and the quaternary monoacrylate. Subsequent ester hydrolysis of the monoacrylate generates the monoquaternary alcohol. In rat plasma, 51W89 is also metabolized by non-specific carboxylesterases to the monoquaternary alcohol and the monoquaternary acid, the former being rapidly hydrolysed further to the more stable acid. It has been reported that laudanosine can be further metabolized via N-dimethylation to yield tetrahydropapaverine. The rate-limiting step in the degradation of 51W89 in human plasma is Hofmann elimination, whilst in rat plasma, the action of non-specific carboxylesterases is rate limiting. As part of the development of 51W89, the disposition of 14C-51W89 following a single intravenous bolus dose was studied in various animal species and humans. In the present work, we describe the identification of 51W89 metabolites in urine and bile from these studies by high performance liquid chromatography/mass spectrometry using pneumatically-assisted electrospray ionization coupled to an on-line radioactivity monitor.(ABSTRACT TRUNCATED AT 250 WORDS)