Differential expression of interleukin-2 by anti-CD3-stimulated peripheral blood mononuclear cells in patients with psoriatic arthritis and patients with cutaneous psoriasis.

The differences in systemic T-cell responses between patients with psoriatic arthritis (PsA) and patients with cutaneous psoriasis (Ps) are still largely unknown. To determine differential features that could be used to distinguish PsA from Ps, we compared the cytokine secretion profile of circulating T cells in patients with PsA, patients with cutaneous Ps and control subjects. We determined Th1, Th2 and Th17 cytokine secretion of anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) using a cytokine bead array. Normality of data distribution was assessed by the Shapiro-Wilk test, and statistical significance was calculated by the Mann-Whitney test. Phenotypic characterization of circulating T cells was performed by fluorescence-activated cell sorting analysis. We found that the major systemic differences distinguishing PsA from cutaneous Ps were the increased secretion of interleukin (IL)-2 by α-CD3-stimulated PBMCs and a higher percentage of circulating CD3+ T cells expressing the proliferation marker CD71 in PsA. These results indicate IL-2 as a possible biomarker of PsA, and suggest a role of circulating T cells with high proliferative capacity in the pathogenesis of PsA.

Successful cyclosporine treatment in a case of amicrobial pustulosis associated with immunological abnormalities.

Amicrobial pustulosis associated with autoimmune diseases (APAD) is a clinical entity which was described only recently and few cases are reported in the literature. This condition is characterized by recurrent acute onset with pustular lesions predominantly involving skin folds, genitals, scalp and external auditory canals of young women. The etiopathogenesis of APAD is unknown and the most effective therapeutic treatment seems to be systemic corticosteroids. We describe the case of a 16-year old female patient suffering from APAD successfully treated with cyclosporine A.

Effect of Efalizumab on neutrophil and monocyte functions in patients with psoriasis.

We evaluated the effect of efalizumab on neutrophil and monocyte functions. The in vitro pre-incubation with efalizumab concentrations similar to those reached during in vivo therapy almost completely saturated CD11a binding sites without affecting the membrane expression of CD11b, CD128a or CD128b. There was a significant reduction in the chemotactic activity of the pre-treated cells toward three different chemo-attractants, whereas their phagocytic capacity and production of oxygen radicals remained unchanged. One month after the administration of efalizumab to five patients with psoriasis (T1) circulating neutrophil counts increased by 34% from pre-therapy (T0) with no change in the number of monocytes. In the same patients the CD11a binding sites on phagocytes were >90% saturated, and there was also a significant down-modulation on neutrophils (44% of T0) and monocytes (63% of T0). In line with in vitro results, efalizumab treatment caused a significant deficiency in the chemotactic properties of neutrophils and monocytes, but no changes in phagocytosis, oxidative burst, production of pro-inflammatory cytokines or the membrane expression of CD11b, CD128a and CD128b. Our findings suggest that neutrophils and monocytes may be among the targets of efalizumab activity in patients with psoriasis.

Disseminated granuloma annulare: efficacy of cyclosporine therapy.

Granuloma annulare is an anatomo-clinical entity that is frequently encountered in everyday dermatological practice. We report our experience regarding 4 patients with disseminated granuloma annulare. Each patient was treated with a cycle of cyclosporine therapy for six weeks. A cycle of systemic cyclosporine therapy was started at a dose of 4 mg/kg/day for four weeks, subsequently reduced by 0.5 mg/kg/day every two weeks. The clinical picture more or less completely resolved within three weeks in all of the patients, and there were no relapses during the dose-tapering period or the following 12 months. Cyclosporine was optimally tolerated by all four patients, none of whom experienced any therapy-related side effects. Cyclosporine is a valid therapeutic option for the treatment of disseminated granuloma annulare, although we recommend its use in a protected hospital environment that facilitates patient monitoring.

Phase I clinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamide copolymer doxorubicin]: first member of a new class of chemotherapeutic agents-drug-polymer conjugates. Cancer Research Campaign Phase I/II Committee.

PK1 comprises doxorubicin covalently bound to N-(2-hydroxypropyl)methacrylamide copolymer by a peptidyl linker. Following cellular uptake via pinocytosis, the linker is cleaved by lysosomal enzymes, allowing intratumoral drug release. Radically altered plasma and tumor pharmacokinetics, compared to free doxorubicin, and significant activity in animal tumors have been demonstrated preclinically. We aimed to determine the maximum tolerated dose, toxicity profile, and pharmacokinetics of PK1 as an i.v. infusion every 3 weeks to patients with refractory or resistant cancers. Altogether, 100 cycles were administered (range, 20-320 mg/m2 doxorubicin-equivalent) to 36 patients (20 males and 16 females) with a mean age of 58.3 years (age range, 34-72 years). The maximum tolerated dose was 320 mg/m2, and the dose-limiting toxicities were febrile neutropenia and mucositis. No congestive cardiac failure was seen despite individual cumulative doses up to 1680 mg/m2. Other anthracycline-like toxicities were attenuated. Pharmacokinetically, PK1 has a distribution t(1/2) of 1.8 h and an elimination t(1/2) averaging 93 h. 131I-labeled PK1 imaging suggests PK1 is taken up by some tumors. Responses (two partial and two minor responses) were seen in four patients with NSCLC, colorectal cancer, and anthracycline-resistant breast cancer. PK1 demonstrated antitumor activity in refractory cancers, no polymer-related toxicity, and proof of principle that polymer-drug conjugation decreases doxorubicin dose-limiting toxicities. The recommended Phase II dose is 280 mg/m2 every 3 weeks. Studies are planned in colorectal, NSCLC, and breast cancer patients.

The dispositional enantioselectivity of indobufen in man.

The plasma pharmacokinetics and urinary elimination of the enantiomers of indobufen (2-[p-(1-oxo-2-isoindolinyl)-phenyl]butyric acid), a novel platelet aggregation inhibitor, have been studied in male healthy volunteers given either the racemic compound or the S-enantiomer (200 mg racemate, 100 mg S-enantiomer). Enantiospecific analysis of indobufen in plasma and urine was achieved by HPLC of its L-leucinamide diastereoisomers. After administration of the racemate, the pharmacokinetic behaviour of the R- and S-enantiomers differed, the plasma levels of the S form declining more rapidly [half-lives = 6.2 hr (S), 8.7 hr (R)]. No substantial differences were observed in terms of plasma level profile of S-indobufen when administered alone and in the racemic mixture. A statistically significant difference between the two enantiomers after administration of the racemate was found in the area under the curve (AUC), peak plasma levels (Cmax) and elimination half-life (t1/2 beta) whereas no statistically significant difference was detected in the time of peak (tmax). When the pharmacokinetic parameters Cmax, AUC, t1/2 beta and tmax of S-indobufen administered alone or as racemate were compared, there were no statistically significant differences between treatments as well as between periods and sequences. The urinary excretion of total S-indobufen (free + glucuronide) and of total R-indobufen after administration of the racemate was essentially the same. No difference was observed either in the urinary excretion of total S-indobufen after administration of the racemate or of the S-enantiomer.

The dispositional enantioselectivity of indobufen in rat and mouse.

The plasma pharmacokinetics and urinary elimination of the enantiomers of indobufen, a novel platelet aggregation inhibitor, have been studied in rats and mice given either the racemic compound or the individual enantiomers (rat 8 mg/kg racemate, 4 mg/kg enantiomers; mouse 25 mg/kg racemate, 12.5 mg/kg enantiomers). Enantiospecific analysis of indobufen in plasma and urine was achieved by HPLC of its L-leucinamide diastereoisomers. In rat, the two enantiomers have very different plasma elimination half lives (S, 3.9 hr; R, 12.2 hr), irrespective of the optical form administered. The plasma concentration-time curves of S-indobufen were identical after racemic or S-indobufen, but the plasma levels of R-indobufen were lower after the R-enantiomer than after the racemate. Urinary recovery of free and conjugated indobufen was less than 3% of the dose, independent of the optical form administered. In the mouse, R-indobufen was cleared from plasma more rapidly than its S-antipode (elimination T1/2 R, 2.5 hr; S, 3.8 hr) but differences were smaller than those seen in the rat. The plasma concentration-time curves of the S-enantiomer were the same after racemic or S-indobufen, but levels of its R-antipode were much lower when it was given alone than after administration of the racemate. The urinary recovery of free and conjugated indobufen also exhibited enantioselectivity, with preferential elimination of the S-enantiomer.

Polymer-bound camptothecin: initial biodistribution and antitumour activity studies.

Camptothecin (CPT) is a potent, antitumour drug acting mainly through inhibition of topoisomerase I during the S-phase of the cell cycle. Despite its impressive antitumour activity, clinical development was halted for unpredictable toxic events. Two soluble N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers were synthesised to contain CPT (5 wt.% and 10 wt.%). CPT was covalently linked at its alpha-hydroxyl group to the polymers through a Gly-Phe-Leu-Gly- spacer. In-vitro, CPT-conjugates were fairly resistant to hydrolysis in plasma as in buffer at neutral pH (0.2-0. 4% free CPT/h), while elastase and cysteine-proteases were able to release the active drug. Plasma levels in mice after intravenous administration of CPT-conjugates confirmed the modest hydrolysis in plasma. Plasma levels were approximately 5-fold lower than those observed at the highest tolerated dose of CPT administered in classical vehicles. Biodistribution in HT29 human colon carcinoma bearing mice was carried out after i.v. injection of [3H]CPT-conjugate and free [3H]CPT. Radioactivity uptake in tumour was evident only after [3H]CPT-conjugate treatment. Repeated intravenous administration of CPT-conjugates to HT29-bearing mice gave more than 90% tumour inhibition, some complete tumour regressions and no toxic deaths. The improved pharmacological profile on HT29 human colon carcinoma xenografts of the first poly(HPMA)-CPT conjugates might be ascribed to their prolonged intra-tumour retention and sustained release of the active drug.

Determination of a new polymer-bound paclitaxel derivative (PNU 166945), free paclitaxel and 7-epipaclitaxel in dog plasma and urine by reversed-phase high-performance liquid chromatography with UV detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of PNU 166945, a new polymer-bound paclitaxel derivative, free paclitaxel and 7-epipaclitaxel in dog plasma and urine has been developed. The method involves a solid-phase extraction of free paclitaxel and its possible degradation product 7-epipaclitaxel from plasma and urine, previously buffered with an equal volume of 0.05 M or 1 M KH2PO4 respectively, on 1-ml cyanopropyl columns. Cartridges elution was performed with the mobile phase, 0.05 M (pH 4.6) monobasic potassium phosphate-acetonitrile mixture (45:55, v/v). The samples were chromatographed on a reversed-phase octyl 4-microns column with UV detection at 229 nm. The retention times of paclitaxel and 7-epipaclitaxel were about 14 and 22 min, respectively. Determination of total paclitaxel (free + polymer-bound) was performed after release of paclitaxel from the polymeric carrier by chemical hydrolysis at room temperature (22 degrees C) for 20 h. After addition of 0.5 ml of methanol-0.1 M KH2PO4 mixture (50:50, v/v, pH = 7.5) to 0.5 ml of plasma or urine, paclitaxel was analysed as described above. PNU 166945 concentration was then determined by subtraction of free from total paclitaxel. The linearity, precision, accuracy and recovery of the method were evaluated. The limit of quantitation of the method was 5 ng/ml for biological fluid for paclitaxel and 7-epipaclitaxel and 20 ng/ml for PNU 166945 (as paclitaxel equivalent).

Sensitive procedure for the determination of reboxetine enantiomers in human plasma by reversed-phase high-performance liquid chromatography with fluorimetric detection after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive and selective high-performance liquid chromatographic method for the determination of reboxetine enantiomers in human plasma was developed. Although two chiral centres are present in reboxetine, its stereospecific synthesis leads to two rather than four possible enantiomers. After extraction from plasma and reaction with (+)-1-(9-fluorenyl)ethyl chloroformate, reboxetine enantiomers were separated as diastereoisomeric derivatives by reversed-phase high-performance liquid chromatography (HPLC) and determined by fluorimetric detection. The HPLC analysis time was about 90 min. The linearity, precision, accuracy and limit of quantification of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was assessed by the analysis of plasma samples obtained from a healthy male volunteer who had received a single oral dose of 4 mg of reboxetine in tablet form.

Determination of PNU-248686A, a novel matrix metalloproteinase inhibitor, in human plasma by liquid chromatography-tandem mass spectrometry, following protein precipitation in the 96-well plate format.

A sensitive, specific and high-throughput analytical method for the quantitation of PNU-248686A (I), in human plasma has been developed. I, sodium (2R)-3-[[(4'-chloro(1,1'-biphenyl)-4-yl]sulfonyl]-2-hydroxy-2-[(phenylsulfanyl)methyl] propanoate, is an orally active matrix metalloproteinase (MMP) inhibitor developed for the treatment of solid tumors over-expressing MMPs. Concentrations of I, as free acid, were determined in human plasma by LC-MS-MS after plasma protein precipitation in the 96-well plate format. Aliquots of plasma (50 microl) were placed into the plates and 0.2 ml of methanol was added. The plates were shaken for 5 min and centrifuged at 1500 g for 10 min. Aliquots of 10 microl of the supernatants were then directly injected into the LC-MS-MS system. A Symmetry Shield C. column (50 x 2.1 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase was 5 mM ammonium formate buffer solution pH 5.0-acetonitrile (60:40. v/v) with a flow-rate of 0.3 ml/min. Retention time of I was about 1.2 min. Total cycle time was 2.5 min. MS detection used the Applied Biosystems-MDS Sciex API 3000 with TurbolonSpray interface and single reaction monitoring (461 --> 251 m/z transition) operated in negative ion mode. Calibration curves were constructed by plotting the area of the compound (y) against its concentration (x). A weighed linear regression (weighting factor 1/x(2)) was used to calculate I concentrations in quality control and unknown samples. The method was fully validated over the range of 5.0-5000 ng/ml. The suitability and robustness of the method for in vivo samples was confirmed by analysis of plasma samples from a pilot clinical study.

Determination of FCE 26644, a new polysulphonated derivative of distamycin A, in monkey plasma by reversed-phase ion-pair high-performance liquid chromatography with ultraviolet detection.

A sensitive and selective ion-pair high-performance liquid chromatographic method for the determination of 7,7'-carbonylbis[imino-N-methyl-4, 2-pyrrolecarbonylimino(N-methyl-4,2-pyrrole)carbonylimino]¿- bis(1,3-naphthalenedisulphonic acid), tetrasodium salt in monkey plasma has been developed. The compound and internal standard (bromphenol blue) were extracted from plasma samples were methylene chloride (twice) after deproteination with acetonitrile and addition of the ion-pairing agent (tetrabutylammonium hydroxide). The combined organic phases were dried, the residue dissolved in the mobile phase and then analysed by reversed-phase ion-pair liquid chromatography under isocratic conditions. The HPLC analysis time was about 20 min. Quantitation was achieved by UV detection at 323 nm. The linearity, precision and accuracy of the method were evaluated. The limit of quantitation was 0.3 microgram/ml plasma. No interference from blank monkey, mouse, rat, dog and human plasma was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from three male cynomolgus monkeys that had received a 20 mg/kg single i.v. dose of the test compound.

Quantitation of SU1 1248, an oral multi-target tyrosine kinase inhibitor, and its metabolite in monkey tissues by liquid chromatograph with tandem mass spectrometry following semi-automated liquid-liquid extraction.

SU11248 is a potent inhibitor of PDGFR, VEGFR, KIT, and Flt3, and is currently under Phase I clinical evaluation as an anticancer drug. A sensitive and specific analytical method for the quantitation of SU11248 and its metabolite in several monkey tissues (liver, kidney, brain and white fat) using LC-MS-MS following semi-automated liquid-liquid extraction (LLE) was developed and validated. Amounts of 50 mg of tissue were homogenized using an ultrasonic processor. After addition of the stable labelled internal standard (IS) and ammonium hydroxide (0.3%), samples were extracted with 2.5 ml of tert-butyl methyl ether. Following centrifugation, aliquots of 1.8 ml of the organic phase were transferred into a 96-well plate. The Packard Multiprobe II robotic liquid handler was used to perform all steps mentioned above. The organic phase was dried and the residue was reconstituted with 800 microl of 15 mM ammonium formate buffer solution (pH 3.25) using a Tomtec Quadra 96 workstation. Aliquots of 10 microl of the resulting solution were injected into the LC-MS-MS system. A Symmetry Shield C8 column (50 mm x 2.1 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase was 15 mM ammonium formate buffer solution (pH 3.25)-acetonitrile (74:26 (v/v)) with a flow-rate of 0.35 ml/min. Retention times of the metabolite and SU11248 were about 2.5 and 3.5 min, respectively. Total cycle time was 5 min. MS detection used the Applied Biosystems-MDS Sciex API 3000 with TurbolonSpray interface and multiple reaction monitoring (MRM) operated in positive ion mode. The method was validated for both compounds over the calibration range of about 2 and 2000 ng/g. The suitability and robustness of the method for in vivo samples were confirmed by analysis of monkey tissues from animals dosed with SU11248.

Association of cyclosporine and 311 nM UVB in the treatment of moderate to severe forms of psoriasis: a new strategic approach.

Psoriasis is a T-lymphocyte mediated autoimmune disease. The response to therapies targeting T-lymphocytes suggest that the latter is a key cell in the pathogenesis of the disease. Cyclosporine (CsA) inhibits the proliferation and the IL-2 dependent expansion of T-lymphocytes. Ultraviolet radiation is an effective treatment for psoriasis. Several studies have demonstrated a significant improvement of the therapeutic response when narrow-band radiation is issued by TL-01 fluorescent lamp compared to broad- band UVB issued by other fluorescent sources. The effects of UVB on the immune system appear to be limited to the cell-mediated compartment of the immune response. In order to reduce the cumulative dose of UVB and limit the toxicity of drugs in the therapy of psoriasis, phototherapy with UVB has been used as treatment in association with other standard therapies. The purpose of the study is to evaluate, in patients with moderate to severe psoriasis a combined therapy with Cyclosporine A and 311 nm UVB phototherapy.

Determination of PNU 153429, a new polysulphonated derivative of distamycin A, in rat plasma by reversed-phase ion-pair high-performance liquid chromatography with ultraviolet detection.

A rapid and selective ion-pair high-performance liquid chromatographic (HPLC) method for the determination of 2,2'-(carbonylbis(imino-N-methyl-4,2-pyrrole carbonylimino (N-methyl-4,2-pyrrole)carbonylimino)) -bis(1,5-naphtalenedisulphonic acid), tetrasodium salt (PNU 153429,I) in rat plasma has been developed. I is a new drug currently under investigation for the treatment of rheumatoid arthritis. Aliquots of 100 microliters of plasma spiked with 10 microliters of internal standard solution (PNU 145156E, I.S.) were added to 100 microliters of acetonitrile and vortex mixed. After centrifugation, diluted aliquots of the supernatant were transferred to autosampler vials and analyzed by reversed-phase ion-pair liquid chromatography under isocratic conditions. The retention times of I.S. and I were approximately equal to 8 and 12 min, respectively. Quantitation was achieved by ultraviolet detection at 323 nm. The assay had a limit of quantitation of 0.1 micrograms ml-1 when 100 microliters of plasma were analyzed. The linearity, precision and accuracy of the method were evaluated. No interference from blank rat, mouse, dog, monkey and human plasma was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from three cannulated male rats that had received a single 100 mg kg-1 i.v. dose of the test compound.

Bioanalytical method validation: a risk-based approach?

Bioanalysis frequently involves the measurement of very low analyte concentrations in complex and potentially variable matrices. It is not possible to test in validation every possible circumstance that may be encountered when analyzing study samples; logically, therefore, some risk of obtaining erroneous results exists when validated methods are applied to study samples. An initial attempt has been made to apply a risk management tool to the bioanalytical situation, with the hope that this will stimulate further discussion on the idea of more formally addressing "risk" with regards to bioanalytical method validation.

LC-MS-MS determination of nemorubicin (methoxymorpholinyldoxorubicin, PNU-152243A) and its 13-OH metabolite (PNU-155051A) in human plasma.

A selective and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for quantitative determination of nemorubicin, (PNU-152243A, 3'-deamino-3'[2(S)-methoxy-4-morpholinyl]doxorubicin) hydrocloride and its reduced metabolite PNU-155051 in human plasma has been developed and validated. The method involved solid phase extraction (SPE) in 96-well plates. Plasma samples (0.5 ml plasma, spiked with doxorubicin as internal standard and diluted with 0.5 ml of 0.01 M borate buffer, pH 8.4) were extracted using Oasis HLB SPE material. The elution of PNU-152243, PNU-155051 and of IS was performed with 1 ml of methanol:0.1 M formic acid mixture (90:10, v/v). The organic phase was reduced to dryness under a stream of nitrogen at 20 degrees C and the residue was reconstituted with 0.25 ml of 10 mM ammonium formate buffer pH 4.15:acetonitrile mixture (90:10, v/v). Aliquots of 60 microl of the resulting solution were injected onto the LC-MS-MS system. A Zorbax SB C18 column (2.1 x 150 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase consisted of ammonium formate buffer 10 mM pH 4.15:acetonitrile (73:27, v/v) with a flow-rate of 0.2 ml/min. Detection was achieved by a PE-SCIEX API 3000 with Turbo IonSpray interface, and multiple reaction monitoring (645 --> 321 for PNU-152243, 647 --> 363 for PNU-155051 and 545 --> 345 m/z for doxorubicin) operated in positive ion mode. A weighted linear regression was used to calculate PNU-152243 and PNU-155051 concentrations in QC and unknown samples. Linearity, precision, accuracy and recovery of the method were evaluated over the concentration range of 0.1-5 ng/ml for both compounds. No interference from blank human plasma was observed. The suitability of the method for in vivo samples was assessed by the analysis of samples obtained from patients who had received a single intrahepatic artery dose of PNU-152243A.

A sensitive procedure for the quantitation of free and N-(2-hydroxypropyl) methacrylamide polymer-bound doxorubicin (PK1) and some of its metabolites, 13-dihydrodoxorubicin, 13-dihydrodoxorubicinone and doxorubicinone, in human plasma and urine by reversed-phase HPLC with fluorimetric detection.

A high-performance liquid chromatographic assay has been developed and validated for the determination in plasma and urine of doxorubicin (DXR) and some of its metabolites released in vivo from an N-(2-hydroxypropyl)methacrylamide (HPMA) polymer containing DXR linked through its aminosugar moiety to the polymer via an oligopeptide spacer (PK1). The method also allows measurement of the DXR still bound to the polymer. Following addition of two internal standards, the free compounds were extracted twice with isopropanol-chloroform (25:75, v/v). The first extraction was performed at physiological pH and the second after buffering at pH 8.4, in order to extract the aglycones and the glycosides, respectively. Determination of total DXR (polymer-bound plus free DXR) was performed, after quantitative acid hydrolysis to release doxorubicinone from free or polymer-bound DXR, by extraction with the same solvent mixture at pH 7.4. In both cases the organic phase was evaporated to dryness; the compounds were then separated by reversed-phase high-performance liquid chromatography (HPLC) under isocratic conditions and quantitated by fluorimetric detection. In the chromatograms all the analytes appeared to be separated at the baseline and no interference from blank human plasma and urine was observed. The suitability of the method for in vivo samples was checked by the analysis of plasma and urine samples obtained from a cancer patient who had received a single intravenous dose of the test compound.

LC-MS-MS determination of exemestane in human plasma with heated nebulizer interface following solid-phase extraction in the 96 well plate format.

A sensitive, specific and rapid analytical method for the quantitation of exemestane (EXE) in human plasma has been developed. EXE, 6-methylen-androsta-1,4-diene-3,17-dione, is an orally active irreversible steroidal aromatase inhibitor used for the therapy of metastatic postmenopausal breast cancer, with estrogen-dependent pathological conditions. The method involves extraction of EXE from human plasma by solid phase extraction using C2 endcapped sorbent in the 96 well plate format (50 mg/2 ml). After conditioning of the sorbent with 1 ml of acetonitrile (x2) the plates were rinsed with 1 ml of water (x2). The prepared samples (0.5 ml plasma, spiked with [13C3] EXE as internal standard (IS) and diluted with 0.5 ml water) were loaded and drawn through the plate with a minimum of vacuum. The plates were then washed with 1 ml acetonitrile:water (10:90) followed by a drying step for 30 min at full vacuum. Elution was by 0.15 ml of 0.1% trifluoracetic acid in acetonitrile (x2) under a minimum of vacuum. Aliquots of 80 microl were finally injected into the LC-MS-MS system. A Zorbax SB C8 column (4.6 x 150 mm, 5 microm) was used to perform the chromatographic separation; the mobile phase was 100% acetonitrile. MS detection used the heated nebulizer interface, with multiple reaction monitoring (MRM) (297-->121 m/z for EXE and 300-->123 m/z for IS) operated in positive ion mode. A weighed linear regression analysis (weighing factor 1/x2) was used to calculate EXE concentration in standard and unknown samples. The method was fully validated in the concentration range 0.05-25 ng ml(-1).