Richard F. Venn

Direct analysis of crude plasma samples by turbulent flow chromatography/tandem mass spectrometry

SummaryTurbulent flow chromatography coupled to tandem mass spectrometry (TFC-MS-MS) has recently emerged as a potentially fast, sensitive and specific technique for the direct analysis of pharmaceutical compounds from crude plasma. TFC-MS-MS removes the need for time-consuming sample preparation procedures such as solid-phase extraction (SPE) or liquid-liquid extraction (LLE). A relatively high flow rate combined with the use, of an HPLC column with large porous particles allows the on-line clean up and quantification of compounds in plasma samples. Until, now, the amount of plasma directly injected into TFC systems has rarely exceeded 30 μL in order to prevent rapid column degradation. Increasing the injection volume also induces high carry-over levels, particularly for drugs with basic and/or lipophilic properties.This paper describes the first genetic TFC-MS-MS method developed in a 96-well format, which allows the direct injection of 200 μL of 1∶1 diluted plasma (equivalent to 100 μL neat plasma). An average, of 390 injections was carried out with each extraction column. More than 2000 dog plasma samples were injected into the system without any sign of carryover. The method was fully validated over a 5–500 ng mL−1 range for three basic compounds: doxazosin, CP122,288 and dofetilide. The imprecision was 1.2 to 8.3% for doxazosin, 1.5 to 4% for CP122,288 and 1.6 to 9.2% for dofetilide. The inaccuracy ranged from 6% to 7.9%. This generic methodology was then used to assay two structurally unrelated development compounds, showing that the method accuracy and sensitivity were adequate for the early pharmacokinetic (PK) studies in animals.

Selective solid phase extraction of a drug lead compound using molecularly imprinted polymers prepared by the target analogue approach

SummaryMolecularly imprinted polymers have been evaluated at the sample clean-up stage in the analysis of a drug lead compound. In order to circumvent quantification problems related to bleeding of the template, a structurally related analogue of the latter was used. This was selected based on criteria related to interaction site location, solubility, availability and stability of the analogue. Selection of suitable polymerisation conditions was then made using a small batch format (ca. 50 mg) and rapid assessment of binding in the equilibrium mode. It was found that the amount of template could be greatly reduced compared to the conventional protocol, requiring only 5 μmol of template per gram of polymer without seriously compromising the performance of the materials for chromatographic or SPE applications.

Fluorescence determination of sulphobutylether-β-cyclodextrin in human plasma by size exclusion chromatography with inclusion complex formation

A selective method for the determination of sulphobutylether-beta-cyclodextrin (SBECD) in human plasma has been developed and validated over the range 4-200 microg ml(-1). SBECD is extracted from plasma using end-capped cyclohexyl solid phase extraction cartridges. This is followed by high performance size exclusion chromatography with a mobile phase consisting of 1-naphthol (0.1 mM) in methanol-potassium nitrate (0.2 M) (1:9 v/v), 1 ml min(-1). The high aqueous content of the mobile phase quenches the fluorescence of 1-naphthol. However, the naphthol forms an inclusion complex with SBECD. The non-polar bucket environment of the inclusion region restores the fluorescence, which is measured at excitation and emission wavelengths of 290 and 360 nm, respectively, when SBECD elutes from the column.

Clinical analysis of sampatrilat, a combined renal endopeptidase and angiotensin-converting enzyme inhibitor I : Assay in plasma of human volunteers by atmospheric-pressure ionisation mass-spectrometry following derivatisation with BF3-methanol

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. A method for plasma was developed and validated employing HPLC APCI MS-MS. The plasma samples were extracted on solid-phase extraction cartridges, derivatised with BF3-methanol, diluted, extracted again and then subjected to HPLC APCI-MS-MS. Derivatisation was necessary because the two carboxyl group in the molecule prevented efficient ionisation in the heated nebuliser source. The calibration range was from 0.5 to 20 ng ml(-1) and the lower limit of quantification was 0.5 ng ml(-1). Imprecision and inaccuracy were determined on three separate occasions at three concentrations (0.5, 5 and 20 ng ml[-1]) and shown to be lower than 10% in every case.

Clinical analysis of sampatrilat, a combined renal endopeptidase and angiotensin-converting enzyme inhibitor: II: Assay in the plasma and urine of human volunteers by dissociation enhanced lanthanide fluorescence immunoassay (DELFIA)

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. An HPLC-atmospheric-pressure chemical ionisation mass-spectrometric (HPLC-APCI-MS-MS) assay had been already validated (R.F. Venn et al., J. Pharm. Biomed. Anal., in press), but due to its low throughput an alternative method was sought. As the molecule is peptide-like and not metabolised, we believed the immunoassay approach was appropriate. Thus we developed an immunoassay for the compound using time-resolved fluorescence as an end-point (DELFIA) with lower limits of quantification of 0.2 ng ml(-1) for the plasma assay and 5 ng ml(-1) for the assay in urine. This assay is a 96-well plate based competitive immunoassay; the end-point is the determination of a (non-radioactive) europium label by time-resolved fluorimetry. Sampatrilat is labelled with chelated europium via isothiocyanate chemistry. The advantage of this assay is its extremely high throughput, allowing rapid analysis of many thousands of samples. The DELFIA method was successfully cross-validated with the HPLC-APCI-MS-MS method.

Determination of a potent urokinase-type plasminogen activator, UK-356,202, in plasma at pg/mL levels using column-switching HPLC and fluorescence detection.

A rapid, sensitive and selective method using column-switching HPLC with fluorescence detection has been developed for the determination of UK-356,202, a potent urokinase-type plasminogen activator, in human plasma. A structural isomer of UK-356,202 is used as an internal standard. The lower limit of quantification is 20 pg/mL and the method is linear over a 100-fold concentration range. UK-356,202 is extracted from plasma simply through the removal of proteins by precipitation with acetonitrile. The HPLC system comprises three columns and the cycle time is 9.5 min per sample. The eluate from the extraction column is heart-cut onto a trace enrichment cartridge which is then back-flushed onto a narrow-bore Supelco ABZ+ Plus analytical column. The method has been used to analyze many thousands of samples from clinical and toxicological studies support. Its ruggedness is demonstrated by the use of a single extraction column for the analysis of over 1200 clinical samples.

Analysis of CP-122,288 at femto-molar levels from human plasma using solid-supported extraction and HPLC column switching

SummaryA sensitive and specific method for the analysis of CP-122,288 in plasma has been developed and validated using column-switching HPLC with fluorescence detection. Solid-phase extraction (SPE) cartridges (Chem-Elut) are used to extract analyte and internal standard from human plasma samples. The extracts are analysed by heart-cutting from one analytical HPLC column to a second column with fluorescence detection using excitation and emission wave-lengths of 225 and 350 nm respectively, after post column UV irradiation at 254 nm. The method has a lower limit of quantification (LLOQ) of 10 pgmL−1 and is linear over a fifty-fold range, up to 500 pg mL−1. An internal standard (CP-122,638) is used which is a close chemical analogue of the compound. This validated method has been used extensively for the analysis of CP-122,288 in plasma samples from clinical trials.

Fluorescence determination of sulphobutylether-β-cyclodextrin sodium in human plasma and urine by size-exclusion chromatography with inclusion complex formation

SummaryHPLC methods with fluorescence detection have been developed for the determination of sulphobutylether-β-cyclodextrin sodium (SBECD), a novel excipient for use with drugs of limited aqueous solubility, in human plasma and urine. SBECD contains no chromophore or fluorophore but can be detected by size-exclusion HPLC using excitation and emission wavelengths of 290 and 360 nm when 1-naphthol is included in the mobile phase. The guest-host inclusion complex SBECD forms with the 1-naphthol restores the quenched fluorescence in the mainly aqueous environment of the mobile phase. Using simple solid phase extraction, recoveries of 41% were obtained using aminopropyl cartridges for urine and of 69% using end-capped cyclohexyl cartridges for plasma. These extracts were analysed using size exclusion HPLC. Both methods were precise, accurate and simple to perform. The working ranges were 4 to 200 μg mL−1 and 50 to 1500 μg mL−1 for plasma and urine respectively. The lower limits of quantification were 4 μg mL−1 for plasma and 50 μg mL−1 for urine. The methods were also selective; no significant interferences were observed in six different sources of plasma or urine, or from a range of likely co-medicants.