Improved one-step solid-phase extraction method for morphine, morphine-3-glucuronide, and morphine-6-glucuronide from plasma and quantitation using high-performance liquid chromatography with electrochemical detection

This communication describes an improved one-step solid-phase extraction method for the recovery of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) from human plasma with reduced coextraction of endogenous plasma constituents, compared to that of the authors' previously reported method. The magnitude of the peak caused by endogenous plasma components in the chromatogram that eluted immediately before the retention time of M3G has been reduced (similar to 80%) significantly (p < 0.01) while achieving high extraction efficiencies for the compounds of interest, viz morphine, M6G, and M3G (93.8 +/- 2.5, 91.7 +/- 1.7, and 93.1 +/- 2.2%, respectively). Furthermore, when the improved solid-phase extraction method was used, the extraction cartridge-derived late-eluting peak (retention time 90 to 100 minutes) reported in our previous method, was no longer present in the plasma extracts. Therefore the combined effect of reducing the recovery of the endogenous components of plasma that chromatographed just before the retention time of M3G and the removal of the late-eluting, extraction cartridge-derived peak has resulted in a decrease in the chromatographic run-time to 20 minutes, thereby increasing the sample throughput by up to 100%.

Solid-phase extraction method with high-performance liquid chromatography and electrochemical detection for the quantitative analysis of oxycodone in human plasma

A sensitive and reproducible solid-phase extraction (SPE) method for the quantification of oxycodone in human plasma was developed. Varian Certify SPE cartridges containing both C-8 and benzoic acid functional groups were the most suitable for the extraction of oxycodone and codeine (internal standard), with consistently high (greater than or equal to 80%) and reproducible recoveries. The elution mobile phase consisted of 1.2 ml of butyl chloride-isopropanol (80:20, v/v) containing 2% ammonia. The quantification limit for oxycodone was 5.3 pmol on-column. Within-day and inter-day coefficients of variation were 1.2% and 6.8% respectively for 284 nM oxycodone and 9.5% and 6.2% respectively for 28.4 nM oxycodone using 0.5-ml plasma aliquots. (C) 1998 Elsevier Science BN. All rights reserved.

Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay

Mycophenolic acid is an immunosuppressant administered as a bioavailable ester, mycophenolate mofetil. The pharmacokinetics of mycophenolic acid have been reported to be variable. Accurate measurement of concentrations of this drug could be important to adjust doses. The aim of this study was to compare the enzyme-multiplied immunoassay technique (EMIT [Dade Behring; San Jose, CA, U.S.A.]) for mycophenolic acid with a high-performance liquid chromatographic (HPLC) assay using samples collected from renal transplant recipients. The HPLC assay used solid phase extraction and a C18 stationary phase with ultraviolet (UV) detection (254 nm). The immunoassay required no manual sample preparation. Plasma samples (n = 102) from seven patients, collected at various times after a dose, were analyzed using both methods. Both assays fulfilled quality-control criteria. Higher concentrations were consistently measured in patient samples when using EMIT. The mean (+/- standard deviation [SD]) bias (EMIT-HPLC) was 1.88 +/- 0.86 mg/L. The differences in concentrations were higher in the middle of a dosage interval, suggesting that a metabolite might have been responsible for overestimation. Measurement of glucuronide concentrations by HPLC demonstrated only a weak correlation between assay differences and glucuronide concentrations. If the crossreacting substance is active, EMIT could provide a superior measure of immunosuppression; if inactive, further work is needed to improve antibody specificity. In conclusion, it was found that EMIT overestimates the concentration of mycophenolic acid in plasma samples from renal transplant recipients compared with HPLC analysis.

Extraction and purification of the zwitterions cylindrospermopsin and deoxycylindrospermopsin from Cylindrospermopsis raciborskii

The hepatotoxin cylindrospermopsin (CYN) has been isolated from the cyanobacterium Cylindrospermopsis raciborskii (C. raci.). Efforts to study this toxin have been hampered by the time-consuming requirement to extract it from cultures of the organism. It is usually extracted from lyophilized cells collected from a laboratory culture. Our preliminary work suggested far more of the toxin is available in solution in the culture media than in the cells collected. We have therefore investigated the use of commercially available solid phase extraction sorbents to extract CYN from culture media in which C. raci. has been grown. A range of reverse phase and ion-exchange sorbents were tested across a range of pHs for their ability to retain CYN without success. Subsequently, graphitized carbon cartridges were found to retain CYN strongly. Elution with 5% formic acid in methanol allowed the CYN to be regained for final purification by HPLC. Deoxy-CYN, an analog of CYN can also be extracted using this procedure. (C) 2001 John Wiley & Sons, Inc.

Thermal stability analysis of organo-silicates, using solid phase microextraction techniques

An analysis of thermal degradation products evolved during the melt processing of organo-layered silicates (OLS) was carried out via the use of a solid phase microextraction (SPME) technique. Two commerical OLSs and one produced in-house were prepared for comparision. The solid phase microextraction technique proved to be a very effective technique for investigating the degradation of the OLS at a specific processing temperature. The results showed that most available OLSs will degrade under typical conditions required for the melt processing of many polymers, including thermoplastic polyurethanes. It is suggested that these degradation products may lead to changes in the structure and properties of the final polymer, particularly in thermoplastic polyurethanes, which seem significantly succeptable to the presence of these products. It is also suggested that many commercially available OLSs are produced in such a way that results in an excess of unbound organic modifier, giving rise to a greater quantity of degradation products. All OLSs where compared and characterised by TGA and GC-MS. (c) 2004 Elsevier B.V. All rights reserved.

Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly(styrene-divinylbenzene) column

A sensitive high-performance liquid chromatographic assay has been developed for measuring plasma concentrations of methotrexate and its major metabolite, 7-hydroxymethotrexate. Methotrexate and metabolite were extracted from plasma using solid-phase extraction. An internal standard, aminopterin was used. Chromatographic separation was achieved using a 15-cm poly(styrene-divinylbenzene) (PRP-1(R)) column. This column is more robust than a silica-based stationary phase. Post column, the eluent was irradiated with UV light, producing fluorescent photolytic degradation products of methotrexate and the metabolite. The excitation and emission wavelengths of fluorescence detection were at 350 and 435 nm, respectively. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.5), with 6% N,N-dimethylformamide and 0.2% of 30% hydrogen peroxide. The absolute recoveries for methotrexate and 7-hydroxymethotrexate were greater than 86%. Precision, expressed as a coefficient of variation (n=6), was

Quantification of free mycophenolic acid by high-performance liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry

To facilitate the investigation of free mycophenolic acid concentrations we developed a high-performance liquid chromatography tandem mass spectrometry method using indomethacin as an internal standard. Free drug was isolated from plasma samples (500 mul) using ultrafiltration, The analytes were extracted from the ultrafiltrate (200 mul) using C-18 solid-phase extraction. Detection was by selected reactant monitoring of mycophenolic acid (m/z 318.9-->190.9) and the internal standard (m/z 356.0-->297.1) with an atmospheric pressure chemical ionisation interface. The total chromatographic analysis time was 12 min. The method was found to be linear over the range investigated, 2.5-200 mug/l (r>0.990, n=6). The relative recovery of the method for the control samples studied (7.5, 40.0 and 150 mug/l) ranged from 95 to 104%. The imprecision of the method, expressed in terms of intra- and inter-day coefficients of variation, was

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 mul) were prepared by protein precipitation, followed by C-18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 mug/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 mug/l, was 100.3-105.4% and less than or equal to7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degreesC for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase 11 and III clinical trials. (C) 2002 Elsevier Science B.V. All rights reserved.

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

Simultaneous determination of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone concentrations in rat serum by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry

An assay using high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry (ESI-MS-MS) was developed for simultaneously determining concentrations of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone, in 50 mul samples of rat serum. Deuterated (d(3)) analogues of each compound were used as internal standards. Samples were treated with acetonitrile to precipitate plasma proteins: acetonitrile was removed from the supernatant by centrifugal evaporation before analysis. Limits of quantitation (ng/ml) and their between-day accuracy and precision (%deviation and %CV) were-morphine, 3.8 (4.3% and 7.6%); morphine-3-glucuronide, 5.0 (4.5% and 2.9%); oxycodone, 4.5 (0.4% and 9.3%); noroxycodone, 5.0 (8.5% and 4.6%). (C) 2004 Elsevier B.V. All rights reserved.

The combined SPE : ToxY-PAM phytotoxicity assay; application and appraisal of a novel biomonitoring tool for the aquatic environment

Mounting concerns regarding the environmental impact of herbicides has meant a growing requirement for accurate, timely information regarding herbicide residue contamination of, in particular, aquatic systems. Conventional methods of detection remain limited in terms of practicality due to high costs of operation and the specialised information that analysis provides. A new phytotoxicity bioassay was trialled for the detection of herbicide residues in filter-purified (Milli-Q) as well as natural waters. The performance of the system, which combines solid-phase extraction (SPE) with the ToxY-PAM dual-channel yield analyser (Heinz Walz GmbH), was tested alongside the traditional method of liquid chromatography-mass spectrometry (LC-MS). The assay methodology was found to be highly sensitive (LOD 0.1 ng L-1 diuron) with good reproducibility. The study showed that the assay protocol is time effective and can be employed for the aquatic screening of herbicide residues in purified as well as natural waters.