High fat diet causes distinct aberrations in the testicular proteome.

Diet has important effects on normal physiology and the potential deleterious effects of high fat diets and obesity on male reproductive health are being increasingly described. We conducted a histological review of the effects of chronic high fat (HF) diet (using a mouse model fed a 45% fat diet for 21 weeks) with a discovery proteomic study to assess for changes in the abundance of proteins in the testis. Mice on a HF diet became obese and developed glucose intolerance. Using mass spectrometry, we identify 102 proteins affected in the testis of obese mice. These included structural proteins important for the blood testis barrier (filamin A, FLNA), proteins involved in oxidative stress responses (spermatogenesis associated 20, SPATA-20) and lipid homoeostasis (sterol regulatory element-binding protein 2, SREBP2 and apolipoprotein A1, APOA1). In addition, an important regulator protein paraspeckle component 1, PSPC-1, which interacts with the androgen receptor was significantly downregulated. Proteomic data was validated using both Western blotting and immunostaining which confirmed and localised protein expression in both mouse and human testis using biopsy specimens. This study focused mainly on the abnormalities that occurred at the protein level and as a result, we have identified several candidate proteins and conducted pathway analysis around the effects of HF diet on the testis providing novel insights not previously described. Some of the identified targets could be targeted therapeutically and future work is directed in this area.

The application of microbore UPLC/oa-TOF-MS and 1H NMR spectroscopy to the metabonomic analysis of rat urine following the intravenous administration of pravastatin.

The metabonomic effects of hepatotoxic doses of pravastatin on the urinary metabolic profiles of female rats have been investigated using ultra performance liquid chromatography (UPLC)-oa-TOF-MS and, independently, by (1)H NMR spectroscopy. UPLC was performed using a 1 mm microbore column packed with 1.7 microm particles. Examination of the data obtained from the individual animals, aided by statistical interpretation of the data, made it possible to identify potential markers for toxicological effects, with both NMR and UPLC-MS analysis highlighting distinct changes in the urinary metabolite profiles. These markers, which included elevated taurine and creatine, as well as bile acids, were consistent with hepatotoxicity in some animals, and this hypothesis was supported by histopathological and clinical chemistry findings. The analytical data from both techniques could be used to define a metabolic "trajectory" as toxicity developed and to provide an explanation for the lack of hepatotoxicity for one of the animals. The two analytical approaches (UPLC-MS and NMR) were found to be complementary whilst the use of a 1mm i.d. x 100 mm column reduced the amount of sample required for analysis to 2 microL, compared with 10 microL for a 2.1mm i.d. x 100 mm column. The 1mm i.d. column also provided increased signal-to-noise without loss of chromatographic efficiency.

N-O glucuronidation: a major human metabolic pathway in the elimination of two novel anti-convulsant drug candidates.

1. The urinary metabolites of the anti-convulsant compound 4-amino-1-(2,6-difluorobenzyl)-1H-1,2,3-triazolo[4,5-c]-pyridine hydrochloride (GI265080) obtained following a single oral dose to man have been detected and quantified relative to each other using 19F-NMR spectroscopy. 2. The human urinary metabolites of GI265080 were isolated using semipreparative HPLC and unequivocally characterized using 1H-NMR spectroscopy, two-dimensional heteronuclear NMR spectroscopy and mass spectrometry. The assignments of the N-(5)-oxide and the N-(5)-O-glucuronide metabolites of GI265080 were further confirmed by independent synthesis. The urinary metabolites obtained following single oral doses to dog and rat have also been isolated and characterized. 3. The human urinary metabolites of GI265080 comprise the N-(5)-oxide, the quaternary N+-(5)-glucuronide, the 7-hydroxy glucuronide and a glucuronide conjugate of the N-(5)-oxide. The N-(5)-O-glucuronide conjugate is a novel species in human metabolism and is a significant route of elimination of GI265080 in man. 4. The urinary metabolites of the potential anti-convulsant GW273293 (6-amino-3-(2,3,5-trichlorophenyl)pyrazin-2-ylamine) obtained following a single oral dose to man have also been isolated and characterized. The formation of a novel N-O-glucuronide was also observed and was shown to constitute a significant route of elimination of GW273293 in man.

Quantitative analysis of pharmaceuticals in biological fluids using high-performance liquid chromatography coupled to mass spectrometry: a review.

1. The development of bio-analysis of drug molecules over the last 10 years is reviewed, focusing on advances in sample preparation, liquid chromatography and detection. 2. Developments have led to improvements in detection sensitivity, enhancements in specificity and increased capacity. 3. Emerging technologies such as monolithic column chromatography and miniaturized chip-based systems are discussed.

Determination of 4-hydroxytamoxifen in mouse plasma in the pg/mL range by gradient capillary liquid chromatography/tandem mass spectrometry.

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.

Mass directed peak selection, an efficient method of drug metabolite identification using directly coupled liquid chromatography-mass spectrometry-nuclear magnetic resonance spectroscopy.

Mass spectrometry (both MS and MS-MS) has been used to determine which eluting chromatography peaks in an LC-MS-nuclear magnetic resonance (NMR) experiment should be selected for extended NMR spectroscopic measurement. This mass directed selection of chromatographic peaks has been applied to test mixtures and urine samples for identification of drug metabolites. It was used to simultaneously determine when drug-related material was eluting and provided molecular mass information on these components. Stop-flow LC-NMR was used to acquire data for structural characterisation of drug-related components. This work further serves to demonstrate the potential of coupling tandem mass spectrometry using an ion trap spectrometer with LC-NMR spectroscopy, to provide an extremely powerful tool in structural elucidation.

Use of directly coupled ion-exchange liquid chromatography-mass spectrometry and liquid chromatography-nuclear magnetic resonance spectroscopy as a strategy for polar metabolite identification.

Ion-exchange LC-MS and LC-NMR have been successfully used to identify a novel N-acetyl metabolite of a highly polar drug candidate [2-(ethanimidoylamino)ethyl]sulfonyl alanine (GW273629) under development as a therapeutic agent. This has been achieved using a simple HPLC method without the need for complicated and time consuming pre- or post-column derivatisation. Ion-exchange chromatography using simple ionic strength buffer and organic solvent mobile phases, as applied here, should be suitable for the analysis of other charged polar species. Optimisation of the system described could result in the development of a rational generic HPLC approach specifically designed for the characterisation of polar drug molecules and their metabolites.

Parallel ultra-high flow rate liquid chromatography with mass spectrometric detection using a multiplex electrospray source for direct, sensitive determination of pharmaceuticals in plasma at extremely high throughput.

Recent years have seen increasing usage of large particle size stationary phases and ultra-high flow rate liquid chromatography/mass spectrometry (LC/MS) for rapid determination of pharmaceuticals in plasma without prior sample preparation. This lack of sample preparation prior to analysis, together with the extremely high throughput of the chromatography, makes the technique extremely attractive to the bioanalyst. Further, the introduction of multiple sprayer interfaces to mass spectrometers provides the potential for even higher throughput. In this paper, we present parallel ultra-high flow rate liquid chromatography using four columns in parallel and a four-way multiple sprayer interface to the mass spectrometer. We have applied this on both the narrow-bore and capillary scale. This technique enables the quantification of drugs from four plasma samples simultaneously, at nanogram per millilitre concentrations, from small aliquots of plasma without sample preparation and with throughputs of up to 120 samples per hour.

Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS.

1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the aromatic ring, and on the ethyl and isopropyl side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not observed in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LC-MS/MS. The relative proportions of fluorine-containing metabolites were determined in animal species by 19F-NMR signal integration. 3. The fluorine atom of the aromatic ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.

Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in dog, cynomolgus monkey and mini-pig.

1. The metabolism of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxalinecarboxylic acid isopropylester (GW420867X) has been investigated following oral administration to dog, cynomolgus monkey and mini-pig. 2. The urinary metabolites were isolated and characterized using semi-preparative HPLC, NMR and LC-MS/MS. The relative proportions of fluorine-containing metabolites were determined for each species by 19F-NMR signal integration. 3. The metabolite profiles for each species were similar, although the proportion of individual components varied, suggesting that similar metabolic pathways are involved in the biotransformation of GW420867X in the species studied. 4. The urinary metabolites indicated that the major routes of biotransformation included hydroxylation and subsequent glucuronic acid conjugation on the aromatic ring, and on the ethyl and isopropyl side chains. A component was observed in mini-pig urine that corresponded to hydroxylation and glucuronidation accompanied by loss of the fluorine atom.

Ultra-high flow rate capillary liquid chromatography with mass spectrometric detection for the direct analysis of pharmaceuticals in plasma at sub-nanogram per millilitre concentrations.

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.

The use of preparative high performance liquid chromatography with tandem mass spectrometric directed fraction collection for the isolation and characterisation of drug metabolites in urine by nuclear magnetic resonance spectroscopy and liquid chromatography/sequential mass spectrometry.

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.

The application of fast gradient capillary liquid chromatography/mass spectrometry to the analysis of pharmaceuticals in biofluids.

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.

Tandem mass spectrometry linked fraction collection for the isolation of drug metabolites from biological matrices.

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.

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.

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.

Use of reduced sorbent bed and disk membrane solid-phase extraction for the analysis of pharmaceutical compounds in biological fluids, with applications in the 96-well format.

Significant improvements in the isolation of pharmaceutical compounds from plasma, serum and urine, have been achieved using ultra low mass sorbent bed and thin disk solid-phase extraction (SPE) material. The use of low sorbent masses or disk SPE material has allowed a significant reduction in solvent usage and extraction times. The reduction in solvent volumes required has allowed elution volumes to be reduced to as low as 30 microl with high and consistent analyte recovery. Several SPE RP-HPLC methods have been developed using these materials, including LC-MS methods. When the chromatographic conditions allow the eluent to be injected directly or injected after dilution with distilled water Empore disks are the extraction media of choice due to the materials low elution volume requirements. When operated in the 96-well microtitre format this micro-extraction provides a very efficient throughput and requires little sample manipulation.

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.

High-performance chromatographic assay for the sulphoxide metabolite of 2'-deoxy-3'-thiacytidine in human urine.

A high-performance liquid chromatographic method is described for the determination in human urine of GI138870X, the sulphoxide metabolite of a novel dideoxynucleoside analogue, 2'-deoxy-3'-thiacytidine (lamivudine). GI138870X was extracted from human urine using Empore SDB RPS solid-phase extraction disks prior to reversed-phase chromatography with UV detection. The method has shown to be valid over the concentration range 0.5-100 micrograms/ml using a 0.5-ml sample volume.