Journal of Chromatography B (v.804, #2)


News Section (N1).

Urinary benzene determination by SPME/GC–MS by C Prado; J Garrido; J.F Periago (255-261).
The urinary excretion of the unmetabolized benzene seems to be a very good index for biomonitoring benzene in occupationally exposed people. The use of solid phase microextraction (SPME) offers important advantages for its determination. Several variables can influence the benzene extraction process. Experimental design methodology was used to estimate the influence of the different variables and to evaluate the simultaneous effect of the more significant variables on the benzene extraction. The results showed that sample temperature, sample volume and their interaction were the more significant factors. A model was found that relates the amount of benzene extracted with the studied variables. The more adequate working conditions were: extraction temperature 15 °C, incubation time 1 min, extraction time 1 min and 2.5 ml of sample volume. The results indicate that this method is capable of providing sensitive and accurate results for the biomonitoring of benzene in urine.
Keywords: Fractional factorial design; Response surface methodology; Benzene;

A simple, rapid and low cost sample preparation method was developed for quantification of docetaxel in mouse plasma by high-performance liquid chromatography/tandem mass spectrometry with paclitaxel as the internal standard. A small volume of plasma (40 μl) and one-step protein precipitation using methanol and acetonitrile (1:1 (v/v)) were used for sample preparation. The calibration curve for docetaxel in mouse plasma was linear over the range 25–2500 nM. The detection limit was 8 nM. The lower limit of quantitation is 25 nM. The intra- and inter-day precisions (CV) of analysis were 9.5 and 9.7% for the low quality control (LQC), 5.5 and 4.9% for the medium quality control (MQC) and 3.9 and 6.3% for the high quality control (HQC), respectively. The accuracy was 102.5% for LQC, 97.9% for MQC and 108.8% for HQC. This assay has now been applied to evaluation of mouse pharmacogenetics and other clinical pharmacology applications.
Keywords: Docetaxel;

New analytical methods based on liquid chromatography with electrospray tandem mass spectrometry (LC–MS/MS) have been developed and validated for assessing the exposure of greenhouse workers to acetamiprid. Both ambient (potential inhalation and dermal exposure) and internal dose (biological monitoring of urine samples) measurements were carried out. Potential inhalation exposure was assessed using Chromosorb 102 cartridges connected to air personal samplers. Potential dermal exposure was estimated by using whole body dosimetry. The measurement of actual exposure was done by analyzing the parent compound in urine samples of the applicators, after a solid-phase extraction (SPE) step. The methods showed a good accuracy (72–92%), precision (2–13%) and lower limits (few μg l−1). The validated approaches have been applied to assess potential and actual exposure of agricultural workers spraying acetamiprid in greenhouses. The results shown the need to wear personal protective equipment (suits) in order to reduce the absorbed dose of acetamiprid.
Keywords: Biomonitoring; Dermal exposure; Acetamiprid;

In the present work, a high-throughput LC/MS/MS method using a Chromolith RP-18 (50  mm×4.6  mm) monolithic column was developed and partially validated for the determination of bupropion (BUP), an anti-depressant drug, and its metabolites, hydroxybupropion and threo-hydrobupropion (TB), in human, mouse, and rat plasma. A modern integrated liquid chromatograph and an LC/MS/MS system with a TurboIonSpray (TIS) interface were used for the positive electrospray selected reaction monitoring (SRM) LC/MS analyses. Spiked control plasma calibration standards and quality control (QC) samples were extracted by semi-automated 96-well liquid–liquid extraction (LLE) using ethyl acetate. A mobile phase consisting of 8 mM ammonium acetate-acetonitrile (55:45, v/v) delivered isocratically at 5 ml/min, and split post-column to 2 ml/min directed to the TIS, provided the optimum conditions for the chromatographic separation of bupropion and its metabolites within 23 s. The isotope-labeled D6-bupropion and D6-hydroxybupropion were used as internal standards. The method was linear over a concentration range of 0.25–200 ng/ml (bupropion and threo-hydrobupropion), and 1.25–1000 ng/ml (hydroxybupropion). The intra- and inter-day assay accuracy and precision were within 15% for all analytes in each of the biological matrices. The monolithic column performance as a function of column backpressure, peak asymmetry, and retention time reproducibility was adequately maintained over 864 extracted plasma injections.
Keywords: Bupropion; Hydroxybupropion; threo-Hydrobupropion;

Determination of MS-275, a novel histone deacetylase inhibitor, in human plasma by liquid chromatography–electrospray mass spectrometry by Kyunghwa Hwang; Milin R Acharya; Edward A Sausville; Suoping Zhai; Eunhee W Woo; Jürgen Venitz; William D Figg; Alex Sparreboom (289-294).
A rapid method was developed for the quantitative determination of the novel histone deacetylase inhibitor, MS-275, in human plasma. Calibration curves were constructed in the range of 1–100 ng/ml, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step protein precipitation with acetonitrile of 0.1 ml samples. The analysis was performed on a column (75  mm ×4.6  mm i.d.) packed with 3.5 μm Phenyl-SB material, using methanol—10 mM ammonium formate (55:45 (v/v)) as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always ≤5.58 and <11.4% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of MS-275 in a cancer patient.
Keywords: MS-275; Histone deacetylase inhibitor;

Immobilized enzyme reactors based upon the flavoenzymes monoamine oxidase A and B by Nektaria Markoglou; Ruth Hsuesh; Irving W Wainer (295-302).
Monoamine oxidase (MAO) catalyzes the oxidative deamination of amines. The enzyme exists in two forms, MAO-A and MAO-B, which differ in substrate specificity and sensitivity to various inhibitors. Membrane fractions containing either expressed MAO-A or MAO-B have been non-covalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The MAO-containing stationary phases were packed into glass columns to create on-line immobilized enzyme reactors (IMERs) that retained the enzymatic activity of the MAO. The resulting MAO-IMERs were coupled through a switching valve to analytical high performance liquid chromatographic columns. The multi-dimensional chromatographic system was used to characterize the MAO-A (MAO-A-IMER) and MAO-B (MAO-B-IMER) forms of the enzyme including the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions as well as the determination of IC50 values. The results of the study demonstrate that the MAO-A-IMER and the MAO-B-IMER can be used for the on-line screening of substances for MAO-A and MAO-B substrate/inhibitor properties.
Keywords: Immobilized enzyme reactors; Flavoenzymes; Monoamine oxidases;

A stereoselective bioanalytical method for the simultaneous quantification of the enantiomers of verapamil and its active main metabolite norverapamil in human plasma has been developed and validated. The samples were analysed by liquid chromatography–electrospray-tandem mass spectrometry (LC–ESI-MS/MS) in the Selected Reaction Monitoring (SRM) mode using a deuterated internal standard. The stationary phase used for the chiral separation was a Chiral-AGP®. The enantiomers of verapamil were selectively detected from those of norverapamil by the mass spectrometer due to different molecular masses, although there was a chromatographic co-elution. Thus, time-consuming procedures like achiral preseparation or chemical derivatisation could be avoided. Higher detection sensitivity than earlier published methods based on fluorescence detection was obtained, although a mobile phase of high water-content and high flow-rate was introduced into the electrospray interface (85% aqueous ammonium acetate pH 7.4+15% acetonitrile at 0.6 ml/min). The enantiomers of verapamil and norverapamil could be quantified at levels down to 50 pg and 60 pg/500 μl plasma sample, respectively, with R.S.D. in the range of 3.6–7.8%. The presented method was successfully applied to an in vivo intestinal absorption and bioavailability study in humans, using the Loc-I-Gut® method.
Keywords: Enantiomer separation; Verapamil; Norverapamil;

Determination of Ochratoxin A in small volumes of human blood serum by G. Köller; U. Rolle-Kampczyk; I. Lehmann; P. Popp; O. Herbarth (313-317).
A new simple and rapid method for analysing Ochratoxin A (OTA) in small volumes of human blood serum using capillary zone electrophoresis coupled to laser-induced fluorescence is described. The clean-up procedure solely consists of a double extraction step. To improve the reproducibility of migration times and quantification, two internal standards were used. The limit of detection was 0.55 ng/ml, with a linear range of 1–100 ng/ml of OTA in spiked human blood serum. The method is used to rapidly screen suspected patients.
Keywords: Ochratoxin A;

A rapid, robust and sensitive method for the extraction and quantitative analysis of serum fluoxetine (FLX) and norfluoxetine (N-FLX) using a solid-phase extraction (SPE) column and high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed and validated. The sample clean-up step was performed by simple micro-disc mixed-mode (non-polar and strong cation exchange (SCX)) SPE cartridges. Separation of analytes and internal standard (IS) clomipramine (CLO) from endogenous matrix interference was achieved using a Waters Symmetry C8 (150  mm×2.1  mm i.d., 5 μm) reversed-phase narrow bore column. The relative retention times were 8.5, 9.6 and 10.5 min for FLX, N-FLX and CLO, respectively with a low isocratic flow rate of 0.3 ml/min. Chromatographic run time was completed in 15 min and peak area ratios of analytes to IS were used for regression analysis of the calibration curve. The latter was linear from 10 to 4000 nmol/l using 0.5 ml sample volume of serum. The average recovery was 95.5% for FLX and 96.9% for N-FLX. The lowest limit of quantitation (LLOQ) for serum FLX and N-FLX was 10 nmol/l (on-column amount of 200 fmol). The method described was used to analyse serum samples obtained from rats given chronic FLX treatment and to examine the relationship between steady state serum drug concentrations and neurochemical changes in several brain regions.
Keywords: Fluoxetine; Norfluoxetine;

Anion-exchange is the most popular chromatography technique in plasmid DNA purification. However, poor resolution of plasmid DNA from RNA often results in the addition of bovine-derived ribonuclease (RNase) A to degrade RNA impurities which raises regulatory concerns for the production of pharmaceutical-grade plasmid DNA. Low capacity for plasmid of most commercial media is another issue affecting the suitability of anion-exchange chromatography for large-scale processing. This study reports the use of anion-exchange chromatography to remove RNA in an RNase-free plasmid purification process. Resolution was achieved through careful selection of adsorbent and operating conditions as well as RNA reduction steps before chromatography. Dynamic capacity for plasmid was significantly increased (to 3.0 mg/ml) so that it is now possible to envisage the large-scale manufacturing of therapeutic-grade plasmid DNA in the absence of added RNase using anion-exchange chromatography as a polishing step.
Keywords: Purification; Plasmid DNA; RNase;

Qualitative studies are described on the metabolism and the toxicological analysis of the nootropic fipexide (FIP) in rat urine using gas chromatography–mass spectrometry (GC–MS). FIP was extensively metabolized to 1-(3,4-methylenedioxybenzyl)piperazine (MDBP), 4-chlorophenoxyacetic acid, 1-[2-(4-chlorophenoxy)acetyl]piperazine, N-(4-hydroxy-3-methoxy-benzyl)piperazine, piperazine, N-(3,4-methylenedioxybenzyl)ethylenediamine, and N-[2-(4-chlorophenoxy)acetyl]ethylenediamine. The authors’ systematic toxicological analysis (STA) procedure using full-scan GC–MS after acid hydrolysis of one urine aliquot, liquid-liquid extraction and acetylation allowed the detection of FIP via its metabolites in rat urine after administration of a common FIP dose. Therefore, this qualitative procedure should also be suitable for detection of a FIP intake in human urine. Differentiation of an intake of FIP from that of other drugs which form common metabolites is discussed.
Keywords: Metabolism; Fipexide;

Quantitation of hydroxyproline in bone by gas chromatography–mass spectrometry by M. Delport; S. Maas; S.W. van der Merwe; J.B. Laurens (345-351).
A validated gas chromatography (GC)–mass spectrometric (MS) method for the analysis of hydroxyproline in rat femur is reported. Hydroxyproline in bone hydrolysates was extracted with an anion exchange resin and the N(O)-tert-butyldimethylsilyl derivatives analyzed by GC–MS. The hydroxyproline concentration was estimated relative to pipecolic acid, 3,4-dehydroproline and n-tetracosane as internal standards. The mass-to-charge ratios (m/z) for the ions used for quantitation by single ion monitoring were 314 m/z for hydroxyproline, 198 m/z for pipecolic acid, 256 m/z for dehydroproline and 57 m/z for n-tetracosane. A coefficient of variation of 5.8% was achieved and the limit of detection was calculated to be 0.233 μmol/l bone hydrolysate.
Keywords: Hydroxyproline;

A fast and sensitive HPLC method using fluorescence detection is developed to quantitate 1-pyrenyldiazomethane (PDAM) derivatized fatty acids derived from the lipid components of both the capsular meningococcal polysaccharide and other impurities such as endotoxin in various meningococcal vaccine samples. The HPLC method is capable of well resolving 13 relevant fatty acids within 40 min by using a multi-stage acetonitrile/water gradient. Endotoxin values measured by HPLC well correlated with results from the standard Limulus amebocyte lysate (LAL) assay. Furthermore, the fatty acid profiles of various process intermediate samples as well as final purified polysaccharide products were determined to better understand and characterize the purification process.
Keywords: Lipid profile; Polysaccharides;

We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC–MS/MS), for the quantification of the novel protease inhibitors (PIs) atazanavir and tipranavir. The sample pre-treatment consisted of protein precipitation with a mixture of methanol and acetronitrile using 100 μl plasma for atazanavir and 50 μl for tipranavir. Chromatographic separation was achieved on an Inertsil ODS3 column (50  mm×2.0  mm i.d., particle size 5 μm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml/min. The analytical run time was 5.5 min. The triple quadrupole mass spectrometer operated in the positive ion-mode and multiple reaction monitoring (MRM) was used for drug quantification. The assay was linear over a concentration range of 0.05–10 μg/ml for atazanavir and 0.1–75 μg/ml for tipranavir. Saquinavir-d5 was used as internal standard. The intra- and inter-day coefficients of variation were less than 3.8% for atazanavir and less than 10.4% for tipranavir. Accuracies were within ±7.3 and ±7.2% for atazanavir and tipranavir, respectively. Both drugs were stable under various relevant storage conditions. The validated concentration ranges proved to be adequate to measure concentrations of human immunodeficiency virus type-1 (HIV-1)-infected individuals. The developed method could easily be combined with a previously developed LC–MS/MS assay for the quantification of protease inhibitors.
Keywords: Atazanavir; Tipranavir;

A gas chromatography–mass spectrometry (GC–MS) assay for the determination of tramadol in human plasma is presented. The synthesis of an N-ethyl analogue of the drug is described and its use as an internal standard for the quantitative measurement of tramadol in human plasma is described. The method involves extraction at plasma pH and analysis of the underivatized drug by gas chromatography–electron ionization mass spectrometry using m/z 58 and 73 for detection of tramadol and internal standard, respectively. The calibration curve was linear in the range of 5–640 ng/ml plasma (r=0.9999). The method was validated in the abovementioned calibration range. Data on solution stability, long- and short-term stability of tramadol in plasma samples, freeze–thaw-stability, as well as inter- and intra-day precision and accuracy have been evaluated and are presented. The application of the method to the pharmacokinetic profiling of the drug is demonstrated.
Keywords: Tramadol; d1-N-Ethyltramadol;

Highly sensitive and specific analytical GC–MS procedures were developed and comprehensively validated for the determination of the opioid narcotics fentanyl, sufentanil and alfentanil and their major nor-metabolites in urine of potentially exposed opioid production workers. A simple, one step extraction protocol was developed using commercially available solid phase extraction (SPE) columns to recover all analytes from urine. The secondary amine functionalities of the nor-metabolites were derivatized to form stable, pentafluorobenzamide (PFBA)-derivatives with good chromatographic properties. Using the penta-deuterated analogues as internal standards, a limit-of-detection (LOD) of 2.5 pg fentanyl/ml, 2.5 pg sufentanil/ml and 7.5 pg alfentanil/ml urine was achieved. For the opioid metabolites the LODs were found to be <50 pg/ml urine. The developed analytical procedures show excellent intra-assay accuracy, particularly considering the ultra low levels of the analytes, with relative errors generally below 10%. Overall, an excellent reproducibility was observed with coefficients of variation below 10% at all spike levels for all opioid parent compounds and their metabolites, except for low norfentanyl concentrations. Upon storage at −30 °C urine samples were found to be stable for at least 2 months as no significant losses of either compound were observed. The developed analytical procedures have been successfully applied in a biological monitoring survey of fentanyl exposed production workers.
Keywords: Occupational exposure; Fentanyl; Sufentanil; Alfentanil;

A simple and highly sensitive high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of lansoprazole enantiomers and their metabolites, 5-hydroxylansoprazole enantiomers and lansoprazole sulfone, in human plasma have been developed. Chromatographic separation was achieved with a Chiral CD-Ph column using a mobile phase of 0.5 M NaClO4–acetonitrile–methanol (6:3:1 (v/v/v)). The analysis required only 100 μl of plasma and involved a solid-phase extraction with Oasis HLB cartridge, with a high extraction recovery (>94.1%) and good selectivity. The lower limit of quantification (LOQ) of this assay was 10 ng/ml for each enantiomer of both lansoprazole and 5-hydroxylansoprazole, and 5 ng/ml for lansoprazole sulfone. The coefficient of variation of inter- and intra-day assay was <8.0% and accuracy was within 8.4% for all analytes (concentration range 10–1000 ng/ml). The linearity of this assay was set between 10 and 1000 ng/ml (r 2>0.999 of the regression line) for each of the five analytes. This method is applicable for accurate and simultaneous monitoring of the plasma levels of lansoprazole enantiomers and their metabolites in the renal transplant recipients.
Keywords: Enantiomer separation; Lansoprazole; 5-Hydroxylansoprazole; Lansoprazole sulfone;

Concentrations of mono(2-ethylhexyl)phthalate (MEHP), and di(2-ethylhexyl)phthalate (DEHP), in serum of healthy volunteers were determined by high performance liquid chromatography (HPLC) with tandem mass spectrometry (LC/MS/MS). The serum was extracted with acetone, followed by hexane extraction under acidic conditions, and then applied to the LC/MS/MS. Recoveries of 20 ng/ml of MEHP and DEHP were 101±5.7 (n=6) and 102±6.5% (n=6), respectively. The limits of quantification (LOQ) of MEHP and DEHP in the method were 5.0 and 14.0 ng/ml, respectively. The concentration of MEHP in the serum was at or less than the LOQ. The concentration of DEHP in the serum was less than the LOQ. Contaminations of MEHP and DEHP from experimental reagents, apparatus and air during the procedure were less than the LOQ and were estimated to be <1.0 and 2.2±0.6 ng/ml, respectively. After subtraction of the contamination, the net concentrations of MEHP and DEHP in the serum were estimated at or <5 and <2 ng/ml, respectively. To decrease contamination by DEHP, the cleanup steps and the apparatus and solvent usage were minimized in the sample preparation procedures. The high selectivity of LC/MS/MS is the key for obtaining reliable experimental data from in the matrix-rich analytical samples and for maintaining a low level contamination of MEHP and DEHP in this experimental system. This method would be a useful tool for the detection of MEHP and DEHP in serum.
Keywords: Di(2-ethylhexyl)phthalate; Mono(2-ethylhexyl)phthalate;

Gas chromatography–mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA) by Dimitrios Tsikas; Anja Mitschke; Frank-Mathias Gutzki; Hartmut H Meyer; Jürgen C Frölich (403-412).
Cytochrome P450 dependent epoxidation and non-enzymic lipid peroxidation of oleic acid (cis-9-octadecenoic acid) result in the formation of cis-9,10-epoxyoctadecanoic acid (cis-EODA). This oleic acid oxide has been identified indirectly in blood and urine of humans. Reliable concentrations of circulating cis-EODA have not been reported thus far. In the present article, we report on the first GC–tandem MS method for the accurate quantitative determination in human plasma of authentic cis-EODA as its pentafluorobenzyl (PFB) ester. cis-[9,10- 2 H 2 ]-EODA (cis-d2-EODA) was synthesized by chemical epoxidation of commercially available cis-[9,10- 2 H 2 ]-9-octadecenoic acid and used as an internal standard for quantification. Endogenous cis-EODA and externally added cis-[9,10- 2 H 2 ]-EODA were isolated from acidified plasma samples (1 ml; pH 4.5) by solvent or solid-phase extraction, converted into their PFB esters, isolated by HPLC and quantified by selected reaction monitoring. The parent ions [M–PFB] at mass-to-charge ratio (m/z) 297 for cis-EODA and m/z 299 for (cis-d2-EODA) were subjected to collisionally-activated dissociation and the corresponding characteristic product ions at m/z 171 and 172 were monitored. In plasma of nine healthy humans (5 females, 4 males), cis-EODA was found to be present at 47.6±7.4 nM (mean±S.D.). Plasma cis-EODA levels were statistically insignificantly different (P=0.10403, t-test) in females (51.1±3.4 nM) and males (43.1±2.2 nM). cis-EODA was identified as a considerable contamination in laboratory plastic ware and found to contribute to endogenous cis-EODA by approximately 2 nM. The present GC–andem MS method should be useful in investigating the physiological role(s) of cis-EODA in humans.
Keywords: cis-9,10-Epoxyoctadecanoic acid; Oleic acid;

A separation using capillary electrophoresis with laser-induced fluorescence (CE-LIF) was applied to the study of green fluorescent protein tagged calmoldulin (GFP-CaM) that was expressed from Escherichia coli and purified with Ni2+-nitrilotriacetate (Ni-NTA) resin column. It was found that GFP-CaM not only has good fluorescence properties under various conditions similar to GFP, but also retains its calcium-binding ability as the native CaM. GFP-CaM was separated and detected by CE-LIF within 10 min with a limit-of-detection (LOD) of 2×10−10  M for an injection volume of 3 nl, higher than that of common chemical fluorescent-tagged protein method. The results indicated that, as a fluorescence probe, GFP could overcome the drawback of inefficient derivatization of chemical fluorescence probes. The interaction between the GFP-CaM and Ca2+ was studied in detail using affinity capillary electrophoresis with laser-induced fluorescence and the dissociation constant (K d) between GFP-CaM and Ca2+ was determined to be 1.2×10−5   M , which is in good agreement with the literature values of untagged CaM (10−6 to 10−5  M) obtained by conventional method. As a preliminary application, the interaction between GFP-CaM and OsCBK was also investigated. The method makes it possible to screen the trace amounts of target proteins in crude extracts interacting with CaM under physiological conditions.
Keywords: Calmodulin; Proteins;

It has been demonstrated that the formation of the hydrophilic metabolites of dexamethasone, 6α- and 6β-hydroxydexamethasone, correlated with cytochrome P450 (CYP) 3A4 enzyme levels. So, the 6β-hydroxydexamethasone/dexamethasone urinary ratio could be a specific marker for human CYP3A4 activity. We have developed a sensitive and specific high-performance liquid chromatographic method for the simultaneous quantification of urinary free dexamethasone and 6β-hydroxydexamethasone using 6α-methylprednisolone as internal standard. This method involved a solid phase extraction of the three compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate (2 ml) followed by diethyl ether (1 ml). Separation of the three analytes was achieved within 24 min using a reversed-phase Nova-Pak C18 analytical column (4 μm, 300  mm×3.9  mm i.d.). An ultraviolet detector operated at 245 nm was used with a linear response observed from 10 to 100 ng/ml for dexamethasone and from 25 to 1000 ng/ml for 6β-hydroxydexamethasone. Obtained from the method validation, inter-assay precision was below 15% and accuracy ranged from 95.7 to 110%. The extraction efficiency of the assay was approximately of 99% and was constant across the calibration range. The lower limit of quantitation was 10 ng/ml for dexamethasone and 25 ng/ml for 6β-hydroxydexamethasone; at these levels, precision was below 16% and accuracy was 99–109%. This method was applied to in vivo measure of the CYP3A4 activity.
Keywords: Dexamethasone; 6β-Hydroxydexamethasone; Cytochrome P450;

Development and validation of a simple liquid chromatographic method with ultraviolet detection for the determination of imatinib in biological samples by Thirumurthy Velpandian; Rajani Mathur; Nitin K Agarwal; Brijesh Arora; Lalit Kumar; Suresh K Gupta (431-434).
The aim of this study was to develop a rapid and sensitive HPLC method with UV detection for the estimation of imatinib from the plasma of patients with chronic myeloid leukemia (CML). The robustness of the method was checked by conducting first dose pharmacokinetics on blood samples from four patients who had been administered Gleevec (100 mg) in an oral dose. Samples were prepared in a simple and single step by precipitating the plasma proteins with methanol and injecting 50 μl aliquot from supernatant was subjected for analysis. Assay was conducted using a C8 column (250  mm×4.6  mm, 5 μm particle size) under isocratic elution with 0.02 M potassium dihydrogen phosphate–acetonitrile (7:3, v/v) at a flow rate of 1 ml/min and detected using photodiode array at 265 nm. Calibration plots in spiked plasma were linear in a concentration range of 0.05–25 μg/ml. The inter and intra-day variation of standard curve was <4% (R.S.D.). This method could be a simple and quick method for the estimation of imatinib from the patient’s plasma.
Keywords: Imatinib;

Validated liquid chromatographic determination of 5-fluorouracil in human plasma by Ibrahim A Alsarra; Mohammed N Alarifi (435-439).
A sensitive, reproducible, selective and accurate high performance liquid chromatographic (HPLC) method for the quantitative determination of 5-flurorouracil in plasma has been developed and validated using isocratic elution and UV detection. The method provides a selective quantifications of 5-flurorouracil without any interference of the endogenous uracil. The assay is performed after a double extraction of 5-flurorouracil and thymine (internal standard) from human plasma using ethyl acetate. The drug and the internal standard were eluted from a Genesis C18 analytical column at ambient temperature with mobile phase consisting of methanol:water (10:90, v/v) adjusted to pH 3.2 with perchloric acid at a flow rate of 1.0 ml/min. The effluent was monitored with an ultraviolet detector at 260 nm. Quantification was achieved by the measurement of the peak-height ratios and the limit of quantification for 5-flurorouracil in plasma was 30 ng/ml. The retention times for 5-flurorouracil, uracil, and thymine were 4.5, 6.0, and 9.0, respectively. The intra-day coefficient of variation (CV) ranged from 1.35 to 4.53% at three different concentrations and the inter-day CVs varied from 1.29 to 4.98%. The relative and absolute recoveries varied from 96 to 101%. Stability tests showed that 5-flurorouracil is stable for at least 72 h in plasma after freezing. The simple method may permit the assessment of 5-flurorouracil plasma concentrations for pharmacokinetic studies in combination with clinical trials.
Keywords: 5-Fluorouracil;

Rapid determination of nimesulide in rabbit aqueous humor by liquid chromatography by Adriana Maltese; Francesco Maugeri; Claudio Bucolo (441-443).
A rapid method was developed for quantification of nimesulide (methanesulfonamide, N-[4-nitro-2-phenoxyphenil]) in rabbit aqueous humor. The analyses were performed by high-performance liquid chromatography using a C18 reversed-phase column (Ultracarb ODS) with UV detection at 300 nm. The mobile phase consisted of acetonitrile-water containing 1% triethylamine (TEA) adjusted to pH 3.2 with orthophosphoric acid. The retention time was 4.5 min. A simple pre-treatment with acetonitrile was used to deproteinize aqueous humor samples. The limit of quantitation was 50 ng/ml. The recovery was over 90%. The relationship between peak areas and concentration was linear over the range between 0.05 and 2.5 μg/ml, with r 2 values over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of nimesulide.
Keywords: Nimesulide;

Author Index (445-447).

Compound Index (449-451).