Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC-UV after solid-phase extraction

An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 microl MeOH/H2O 50/50. A 40 microl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 microg/ml, with a lower limit of quantification of 0.125 microg/ml. The mean absolute recovery of TPV is 77.1+/-4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2-3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.     

UPLC-MS/MS quantification of nanoformulated ritonavir, indinavir, atazanavir, and efavirenz in mouse serum and tissues

Animal pharmacokinetic and tissue distribution assays of antiretroviral therapeutic drugs require accurate drug quantification in biological fluids and tissues. Here we report a simple, rapid, and sensitive ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of commonly used antiretroviral drugs ritonavir (RTV), indinavir (IDV), atazanavir (ATV), and efavirenz (EFV) in mouse serum and tissues (liver, kidney, lung, and spleen). These antiretroviral drugs are currently the cornerstones of common therapeutic regimens for human immunodeficiency virus (HIV) infection. Chromatographic separation was achieved using a gradient mobile phase (5% acetonitrile in methanol and 7.5mM ammonium acetate (pH 4.0)) on an ACQUITY UPLC(?)BEH Shield RP 18 column. All compounds eluted within a 7 min run time. Lopinavir was used as an internal standard. Detection was achieved by dual positive and negative ionization modes on a quadrupole linear ion trap hybrid mass spectrometer with an electrospray ionization (ESI) source. The dynamic range was 0.2-1000 ng/mL for RTV, IDV, and ATV, and 0.5-1000 for EFV. The method was validated and showed high and consistent intra-day and inter-day accuracy and precision for all analytes. This method is used to support the preclinical development studies of targeted- and sustained-release combination ART (nanoART). The current data demonstrate a 1.5-4 fold increase in serum and tissue AUC of nanoformulated ATV, RTV, and EFV administered to mice when compared to native drug. In addition, the tested formulation enhanced exposure of the same anti-HIV drugs in mouse tissues.     

Determination of lopinavir and nevirapine by high-performance liquid chromatography after solid-phase extraction: application for the assessment of their transplacental passage at delivery

An adaptation of the HPLC method previously described for the simultaneous assay of amprenavir, ritonavir, indinavir, saquinavir, nelfinavir and efavirenz after solid-phase extraction is proposed here for the separate analysis of the newer PI lopinavir (LPV) and the NNRTI nevirapine (NVP). After viral inactivation by heat (60 degrees C for 60 min), plasma (600 microl), with clozapine added as internal standard, is diluted 1+1 with phosphate buffer pH 7 and subjected to a solid-phase extraction on a C(18) cartridge. Matrix components are eliminated with 2 x 500 microl of a solution of 0.1% H(3)PO(4) neutralised with NaOH to pH 7. LPV and NVP are eluted with 3 x 500 microl MeOH. The resulting eluate is evaporated under nitrogen at room temperature and is reconstituted in 100 microl MeOH 50%. A 40-microl volume is injected onto a Nucleosil 100, 5 microm C(18) AB column. LPV and NVP are analysed separately using a gradient elution program with solvents constituted of MeCN and phosphate buffer adjusted to pH 5.07 and containing 0.02% sodium heptanesulfonate. LPV and NVP are detected by UV at 201 and 282 nm, respectively. The calibration curves are linear up to 10 microg/ml. The mean absolute recovery of LPV and NVP is 91% and 88%, respectively. The method is precise with mean inter-day C.V.s within 2.1-6.6% and 0.9-1.7% for LPV and NVP, and accurate (range of inter-day deviations -1.1 to +2.4%, and -1.9 to +0.8%, for LPV and NVP, respectively). The method has been validated and is currently applied to the monitoring of LPV and NVP in HIV patients, and has been notably applied in a study aimed at assessing the extent of transplacental passage of nevirapine and PIs, notably lopinavir, at the time of delivery in pregnant HIV-infected women.     

Effect of HIV protease inhibitors on New World Leishmania

The incidence of HIV/Leishmania co-infection decreases after antiretroviral drug therapy; therefore, the in vitro and in vivo activity of three antiretroviral drugs against Leishmania (Viannia) braziliensis and L. (L.) amazonensis was evaluated. Different concentrations of indinavir (IDV), atazanavir (ATV), and ritonavir (RTV) were added to promastigote cultures, and the 50% inhibitory concentration (IC50) was determined. IDV and RTV were also evaluated against intracellular amastigotes, and the Infection Index determined. BALB/c mice, infected with L. (L.) amazonensis in the left footpad, were treated orally with IDV and RTV for 30days, and monitored by measuring the footpad thickness and parasite load of regional lymph nodes and spleen. For promastigotes, IDV exhibited an IC50 value of 100μM against L.(L.) amazonensis. The RTV IC50 for L. (L.) amazonensis and L. (V.) braziliensis were 40 and 2.3μM, respectively, and the ATV IC50 for L. (V.) braziliensis was 266μM. For intracellular amastigotes, IDV (25, 50, and 100μM) significantly decreased the Infection Index of L. (L.) amazonensis (56.8%, 47.9%, and 65.0%) and L. (V.) braziliensis (37.8%, 48.7%, and 43.2%). RTV (12.5, 25, and 50μM) decreased the infection index of L. (L.) amazonensis by 26.3%, 42.4%, and 44.0%, and that of L. (V.) braziliensis by 27.6%, 37.3%, and 39.2%. Antiretroviral-treated mice had a significant reduction in footpad thickness after the third week of IDV and after the fifth week of RTV treatment. However, there was no reduction in parasite load. These results suggest that IDV and RTV have anti-Leishmania activity, but only in higher concentrations.     

Interactions of different inhibitors with active-site aspartyl residues of HIV-1 protease and possible relevance to pepsin

The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors [symmetric cyclic urea inhibitor DMP323, nonhydrolyzable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)] was assessed by differential scanning calorimetry. DeltaT(m) values, defined as the difference in T(m) for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB, and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 degrees C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by DeltaT(m) ratios [DeltaT(m)(PR)/DeltaT(m)(PR(D25N))] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (DeltaT(m) ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (DeltaT(m)(PR)/DeltaT(m)(PR(D29N)) > or = 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (DeltaT(m) ratios of 1.4-2.2). Of the nine FDA-approved clinical HIV-1 protease inhibitors screened, APV, RTV, and DRV competitively inhibit porcine pepsin with K(i) values of 0.3, 0.6, and 2.14 microM, respectively. DSC results were consistent with this relatively weak binding of APV (DeltaT(m) 2.7 degrees C) compared with the tight binding of Ac-PEP (DeltaT(m) > or = 17 degrees C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32, and Ser219 in pepsin, equivalent to Asp25, Asp25', and Asp29 in PR in the binding and stabilization of the pepsin/APV complex.     

A highly sensitive ultra performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) technique for quantitation of protein free and bound efavirenz (EFV) in human seminal and blood plasma

A combined UPLC-tandem mass spectrometric (UPLC-MS/MS) technique has been validated for quantitation of protein free efavirenz (EFV) as well as total concentrations of EFV in human blood and seminal plasma. The analytical method possesses capabilities for concentration measurements of EFV ranging from 0.5 to 10,000ng/ml with an accuracy (%dev) of -5.2-8.0% and precision (%CV) of <8%. Standard curves were linear with coefficients of variation (r(2)) >0.98. The method employs a racemic fluorinated analog of EFV (F-EFV) as the internal standard. EFV and F-EFV were eluted from a reverse-phase UPLC column via gradient elution with detection via negative ion multiple reaction monitoring (MRM). EFV and F-EFV, respectively, were detected via the following MRM transitions: m/z 314.0>244.1 and m/z 298.0>227.9. The time required for the analysis of each sample was 8.0min. The analytical technique is capable of a reliable detection limit of ～15-20fmol of EFV injected on column.     

Select HIV protease inhibitors alter bone and fat metabolism ex vivo

Human immunodeficiency virus (HIV) therapies have been associated with alterations in fat metabolism and bone mineral density. This study examined the effects of HIV protease inhibitors (PIs) on bone resorption, bone formation, and adipocyte differentiation using ex vivo cultured osteoclasts, osteoblasts, and adipocytes, respectively. Osteoclast activity, measured using a rat neonatal calvaria assay, increased in the presence of nelfinavir (NFV; 47.2%, p = 0.001), indinavir (34.6%, p = 0.001), saquinavir (24.3%, p = 0.001), or ritonavir (18%, p < 0.01). In contrast, lopinavir (LPV) and amprenavir did not increase osteoclast activity. In human mesenchymal stem cells (hMSCs), the PIs LPV and NFV decreased osteoblast alkaline phosphatase enzyme activity and gene expression significantly (p < 0.05). LPV and NFV diminished calcium deposition and osteoprotegrin expression (p < 0.05), whereas the other PIs investigated did not. Adipogenesis of hMSCs was strongly inhibited by saquinavir and NFV (>50%, p < 0.001) and moderately inhibited by ritonavir and LPV (>40%, p < 0.01). Expression of diacylglycerol transferase, a marker of adipocyte differentiation, decreased in hMSCs treated with NFV. Amprenavir and indinavir did not affect adipogenesis or lipolysis. These results suggest that bone and fat formation in hMSCs of bone marrow may be coordinately down-regulated by some but not all PIs.     

Simultaneous quantification of lopinavir and ritonavir in human plasma by high performance liquid chromatography coupled with UV detection

High performance liquid chromatography was coupled with UV detection for simultaneous quantification of lopinavir (LPV) and ritonavir (RTV) in human plasma. This assay was sensitive, accurate and simple, and only used 200 μL of plasma sample. Samples were liquid-liquid extracted, and diazepam was used as an internal standard. The chromatographic separation was achieved on a C18 reversed-phase analytic column with a mobile phase of acetonitrile-sodium dihydrogen phosphate buffer (10 mmol L⁻¹, pH 4.80) (60:40, v/v). UV detection was conducted at 205 nm and the column oven was set at 40°C. Calibration curves were constructed between 0.5–20 μg mL⁻¹ for LPV and 0.05–5 μg mL⁻¹ for RTV. The relative standard deviations were 2.16%–3.20% for LPV and 2.12%–2.60% for RTV for intra-day analysis, and 2.34%–4.04% for LPV and 0.31%–4.94% for RTV for inter-day analysis. The accuracy was within 100%±10%. The mean extraction recoveries were 79.17%, 52.26% and 91.35% for RTV, LPV and diazepam, respectively. This method was successfully applied to human plasma samples from patients orally administered a salvage regimen of lopinavir-ritonavir tablets.     

High throughput LC-MS/MS method for simultaneous quantification of lamivudine, stavudine and nevirapine in human plasma

A selective and high throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated to separate, detect and simultaneously quantify lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma using metaxalone as internal standard (IS). After solid phase extraction (SPE), the analytes and the IS were chromatographed on a Symmetry C18 (150 mmx3.9 mm i.d., 5 microm particle size) column using 5 microL injection volume with a run time of 4.5 min. An isocratic mobile phase consisting of 0.5% glacial acetic acid in water:acetonitrile (20:80, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The method was validated over the range of 25-3000 ng/mL for 3TC, 20-2000 ng/mL for d4T and 50-5000 ng/mL for NVP. The absolute recoveries for analytes (>or=86%) and IS (98.12%) achieved from spiked plasma samples were consistent and reproducible. Inter-batch and intra-batch precision (%CV) across four validation runs (LLOQ, LQC, MQC and HQC) was less than 10. The accuracy determined at these levels was within +/-8% in terms of relative error. The method was successfully applied to a pivotal bioequivalence study of [60 (3TC)+12 (d4T)+100 (NVP)] mg dispersible tablets in 60 healthy human subjects under fasting condition.     

Measurement of the anticancer agent gemcitabine and its deaminated metabolite at low concentrations in human plasma by liquid chromatography-mass spectrometry

A liquid chromatography/mass spectrometry (LC-MS) method has been developed and validated for the determination of the anticancer agent gemcitabine (dFdC) and its metabolite 2',2'-difluoro-2'-deoxyuridine (dFdU) in human plasma. An Oasis((R)) HLB solid phase extraction cartridge was used for plasma sample preparation. Separation of the analytes was achieved with a YMC ODS-AQ (5 microm, 120A, [Formula: see text] mm) column. The initial composition of the mobile phase was 2% methanol/98% 5mM ammonium acetate at pH 6.8 (v/v), and the flow rate was 0.2 ml/min. An isocratic gradient was used for 3min, followed by a linear gradient over 4 min to 30% methanol/70% 5mM ammonium acetate at pH 6.8. The gradient returned to the initial conditions over 2 min and remained there for 6 min. The retention times of dFdC, dFdU, and the internal standard 5'-deoxy-5-fluorouridine (5'-DFUR) were 11.46, 12.63, and 13.58 min. The mass spectrometer was operated under negative electrospray ionization conditions. Single-ion-monitoring (SIM) mode was used for analyte quantitation at m/z 262 for [dFdC-H](-), m/z 263 for [dFdU-H](-), and m/z 245 for [5'-DFUR-H](-). The average recoveries for dFdC, dFdU, and 5'-DFUR were 88.4, 84.6, and 99.3%, respectively. The linear calibration ranges were 5-1000 ng/ml for dFdC, and 5-5000 ng/ml for dFdU. The intra- and inter-assay precisions (%CV) were 相似文献   

Validation and clinical application of a high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of 10 anti-retrovirals in human peripheral blood mononuclear cells

This paper reports the validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method that allows the quantification of 10 antiretroviral (ARV) drugs in peripheral blood mononuclear cells (PBMCs) using 6 different isotopic internal standards (IS) and its clinical application. PBMCs are isolated from blood by density gradient centrifugation and drugs are extracted with a 60% methanol (MeOH) solution containing the 6 IS. The cell extract is then injected in the HPLC system and analytes are separated on a Symmetry Shield RP18 2.1 mm × 50 mm column. The different molecules are then detected by MS/MS in electrospray positive or negative ionisation modes and data are recorded using the multiple reaction monitoring (MRM) mode. Calibration curves are constructed in the range of 0.25–125 ng/ml of cell extract by a 1/x² weighted quadratic regression. The regression coefficients obtained are always greater than 0.99 and back calculated values always comprised in the range of ±15% from their nominal concentration. Mean extraction recoveries are greater than 80% for all analytes and the method is accurate and precise with CV and bias lower than 9.4%. The lower limits of quantification (LLOQ) of the different drugs range from 0.0125 to 0.2 ng/ml of cell extract. This method was successfully applied to a cohort of 98 HIV-infected patients treated with Kaletra^® (400/100 mg of lopinavir/ritonavir (LPV/RTV) twice a day, n = 48) or with Stocrin^® (600 mg once a day, n = 50) and has been tested for cellular quantification of tipranavir (TPV) in 2 patients treated with Aptivus^® (500 mg twice a day). The patients treated by Kaletra^® showed mean cell-associated concentrations (CC) of 1819.0 and 917.2 ng/ml, for LPV and RTV, respectively. Patients treated with Stocrin^® showed mean CC of 2388.11 ng/ml while both patients under Aptivus^® showed TPV CC of 4322.7 and 1078.0 ng/ml, respectively. This method can be used to analyze ARV drug concentrations within the target tissue.     

A LC–tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir,maraviroc, darunavir,and etravirine

Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100 μl) is performed by protein precipitation using 600 μl of acetonitrile, after the addition of 100 μl darunavir-d₉ (DRV-d₉) at 1000 ng/ml in MeOH/H₂O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10 min, vortex-mixed again and centrifuged. An aliquot of supernatant (150 μl) is diluted 1:1 with a mixture of 20 mM ammonium acetate/MeOH 40/60 and 10 μl is injected onto a 2.1 × 50 mm Waters Atlantis?-dC18 3 μm analytical column. Chromatographic separations are performed using a gradient program with 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000 ng/ml, 2.5 to 1000 ng/ml, 25 to 10,000 ng/ml, 10 to 4000 ng/ml, and 5 to 2000 ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1–9.8%, and accurate (range of inter-day deviation from nominal values ?3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir–glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.     

Comparative analysis of ER stress response into HIV protease inhibitors: Lopinavir but not darunavir induces potent ER stress response via ROS/JNK pathway

HIV protease inhibitor (PI)-induced ER stress has been associated with adverse effects. Although it is a serious clinical problem for HIV/AIDS patients, comparative analyses of ER stress induction by clinically used PIs have rarely been done. Especially, there is no report on the differential ER stress response between lopinavir (LPV) and darunavir (DRV), although these PIs are the most clinically used PIs. We show here that LPV induces the most potent CHOP expression, ER stress marker, among the 9 Food and Drug Administration (FDA)-approved PIs in human peripheral blood mononuclear cells, several human epithelial cells, and mouse embryonic fibroblasts. LPV induced the most potent ROS production and JNK activation in 9 PIs. A comparison among the most clinically used PIs, ritonavir (RTV), LPV, and DRV, revealed that LPV potently and RTV moderately but not DRV induced ER stress via ROS-dependent JNK activation rather than proteasome inhibition. Finally, we analyzed ER stress induction in tissues of mice intraperitoneally injected with RTV, LPV, and DRV. RTV and LPV but not DRV showed ER stress induction in several mice tissues. In conclusion, we first identify LPV as the most potent ER stress inducing PI among 9 FDA-approved PIs in human cells, and although clinical verification is necessary, we show here that DRV has the advantage of less ROS and ER stress induction potential compared with LPV in vitro and in vivo.     

Simultaneous determination of nimesulide and hydroxynimesulide in rat plasma,cerebrospinal fluid and brain by liquid chromatography using solid-phase extraction

A liquid chromatographic method with UV detection for the quantification of nimesulide (N) and hydroxynimesulide (M1) in rat plasma, cerebrospinal fluid (CSF) and brain tissue is reported. Plasma samples (250 microl) and brain homogenates added with the right amount of the internal standard (I.S., 2'-(cyclohexyloxy)-4'-nitrophenyl methanesulphonanilide, NS398) are extracted on C(18) disposable cartridges by solid-phase extraction (SPE), while CSF samples are analyzed without any extraction. The separation is performed at room temperature on a Waters Symmetry C(18) 3.5 microm (150x4.6 mm I.D.) column with acetonitrile-sodium citrate buffer pH 3.00 (53:47, v/v) as mobile phase, at a flow-rate of 1.1 ml/min and detection at 240 nm. The retention times are 3.3, 6.0 and 9.9 min for M1, N and I.S., respectively. The lower limits of quantitation for either nimesulide and M1 are 25 ng/ml for plasma, 20 ng/ml for CSF and 25 ng/g for brain tissue. The calibration curves are linear up to 10,000 ng/ml for plasma, 5000 ng/ml for CSF and 5000 ng/g for brain tissue. This new assay can be applied to the study of the role of nimesulide in the modulation of neuroinflammatory processes.     

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

Clinical inhibitor amprenavir (APV) is less effective on HIV‐2 protease (PR₂) than on HIV‐1 protease (PR₁). We solved the crystal structure of PR₂ with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR₁ mutant (PR_1M) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR₂. PR_1M more closely resembled PR₂ than PR₁ in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR_1M with APV, DRV, and SQV were compared with available PR₁ and PR₂ complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR_1M and PR₁, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR_1M. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR_1M and PR₂ relative to the strong hydrogen bonds observed in PR₁, consistent with 15‐ and 19‐fold weaker inhibition, respectively. Overall, PR_1M partially replicates the specificity of PR₂ and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV‐2.     

High-performance liquid chromatographic method to screen and quantitate seven selective serotonin reuptake inhibitors in human serum

A high-performance liquid chromatographic screening method (HPLC) is described for the determination of seven selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, milnacipran, paroxetine, sertraline, fluoxetine, citalopram, venlafaxine) and for three pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram and norfluoxetine). A tricyclic antidepressant, clomipramine, was used as an internal standard. The method consists of liquid extraction of serum after alcalinisation at pH 9.50, followed by chromatography on a Beckman C₁₈ reversed-phase column. Compounds were detected at 200.4 nm. The standard curves were linear over a working range of 50–1000 ng/ml for fluvoxamine, 15–1000 ng/ml for fluoxetine, 25–500 ng/ml for norfluoxetine, 50–500 ng/ml for sertraline, 20–500 ng/ml for paroxetine, 25–550 ng/ml for citalopram, 25–750 ng/ml for desmethylcitalopram, 25–800 ng/ml for didesmethylcitalopram, 25–650 ng/ml for milnacipran, and 25–500 ng/ml for venlafaxine. The quantitation limits of the method were 15 ng/ml for fluoxetine, 20 ng/ml for paroxetine, 25 ng/ml for venlafaxine, norfluoxetine and citalopram, and its metabolites, 40 ng/ml for sertraline and 50 ng/ml for fluvoxamine. No interferences were noted with this sensitive and specific method which can be used for therapeutic drug monitoring.     

Enantioselective analysis of propafenone in plasma using a polysaccharide-based chiral stationary phase under reversed-phase conditions

We present a method for the enantioselective analysis of propafenone in human plasma for application in clinical pharmacokinetic studies. Propafenone enantiomers were resolved on a 10-μm Chiralcel OD-R column (250×4.6 mm I.D.) after solid-phase extraction using disposable solid-phase extraction tubes (RP-18). The mobile phase used for the resolution of propafenone enantiomers and the internal standard propranolol was 0.25 M sodium perchlorate (pH 4.0)–acetonitrile (60:40, v/v), at a flow-rate of 0.7 ml/min. The method showed a mean recovery of 99.9% for (S)-propafenone and 100.5% for (R)-propafenone, and the coefficients of variation obtained in the precision and accuracy study were below 10%. The proposed method presented quantitation limits of 25 ng/ml and was linear up to a concentration of 5000 ng/ml of each enantiomer.     

High-performance capillary electrophoresis measurement of dolastatin-10

A high-performance capillary electrophoresis (HPCE) assay was used to determine the concentration of a potent cytotoxic agent, dolastatin-10, in human plasma. Following extraction from plasma, using a solid-phase C₁₈ cartridge, capillary zone electrophoresis was used to separate, detect and quantitate dolastatin-10 using the structurally related compound dolastatin-15 as the internal standard. Migration times for both dolastatins are less than 20 min. The recovery of the drug was approximately 90% and was quantified over the assay range of 39 to 5000 ng/ml with good precision and accuracy. The method is linear up to 5000 ng/ml with a lower limit of detection of 25 ng/ml. Data resulting from the use of the assay for the in vitro metabolism of the drug are presented. This is the first report of a validated HPCE assay for determining dolastatin-10 levels in human plasma.