Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography–tandem mass spectrometry

A column-switching liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 μl) were injected onto a C₁₈-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0–acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C₁₈ column, with a mixture of 20 mM ammonium acetate–acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M−H]⁻ were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M−H]⁻ in MS–MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M−H−Glu]⁻, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025–100 μg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78–103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.     

Metabolic profiling of valproic acid by cDNA-expressed human cytochrome P450 enzymes using negative-ion chemical ionization gas chromatography–mass spectrometry

A sensitive negative ion chemical ionization (NCI) gas chromatographic–mass spectrometric (GC–MS) method was modified for the quantitation of valproic acid (VPA) metabolites generated from in vitro cDNA-expressed human microsomal cytochrome P450 incubations. The use of the inherent soft ionization nature of electron-capture NCI to achieve high sensitivity enabled us to conduct kinetic studies using small amounts of recombinant human P450 enzymes. The assay is based on the selective ion monitoring of the intense [M−181] fragments of pentafluorobenzyl (PFB) esters in the NCI mode, and has the following features: (1) a micro-extraction procedure to isolate VPA metabolites from small incubation volumes (100 μl); (2) a second step derivatization with tert.-butyldimethylsilylating reagents to enhance sensitivity for hydroxylated metabolites; (3) a short run-time (<30 min) while maintaining full separation of 15 VPA metabolites by using a narrow-bore non-polar DB-1 column plus a new temperature gradient; and (4) good reproducibility and accuracy (intra- and inter-assay RSDs <15%, bias <15%) by using seven deuterated derivatives of analytes as internal standards. The derivatives of mono- and diunsaturated metabolites, like the parent drug, produced abundant [M−181]⁻ ions while the hydroxylated metabolites gave an ion at m/z of 273, corresponding to the [M−181]⁻ ion of the tert.-butyldimethylsilyl ethers. In conclusion, the GC–NCI-MS analysis of valproate metabolites provided us with a high resolution and sensitivity necessary to conduct metabolic and kinetic studies of valproic acid in small volume samples typical of the in vitro cDNA-expressed micro-incubation enzymatic systems.     

Modified gas chromatographic–mass spectrometric assay for the determination of gacyclidine enantiomers in human plasma

A modified method for the determination of gacyclidine enantiomers in human plasma by GC–MS with selected-ion monitoring using the deuterated derivative of gacyclidine (d₃-gacyclidine) as internal standard was developed. Following a single-step liquid–liquid extraction with hexane, drug enantiomers were separated on a chiral fused-silica capillary column (CP-Chirasil-Dex; Chrompack). The fragment ion, m/z 266, was selected for monitoring d₃-gacyclidine (retention times of 35.2 and 35.6 min for the (+)- and (−)-enantiomer, respectively) whereas the fragment ion, m/z 263, was selected for quantitation of gacyclidine (retention times of 35.4 and 35.9 min for the (+)- and (−)-enantiomer, respectively). The limit of quantitation for each enantiomer was 0.3 ng/ml, using 1 ml of sample, with a relative standard deviation (RSD) <14% and a signal-to-noise ratio of 5. The extraction recovery of both gacyclidine enantiomers from human plasma was about 75%. The calibration curves were linear (r²>0.996) over the working range of 0.312 to 20 ng/ml. Within- and between-day RSD were <9% at 5, 10 and 20 ng/ml, and <16% at 0.312, 0.625, 1.25 and 2.5 ng/ml. Intraday and interday bias were less than 11% for both enantiomers. The chromatographic behavior of d₃-gacyclidine remained satisfactory even after more than 500 injections. Applicability of this specific and stereoselective assay is demonstrated for a clinical pharmacokinetic study with racemic gacyclidine.     

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography–mass spectrometry

We report here the development and validation of an LC–MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C₁₈ reversed-phase column using an acetonitrile–water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS–MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477→266), DMLOP (m/z 463→252) and A-LOP (m/z 519→266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3±1.50% (range: 70.7–73.7%) and 79.4±12.8% (64.9–88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.     

Toxic Alexandrium minutum (Dinophyceae) from Vietnam with new gonyautoxin analogue

Clonal cultures of Alexandrium species collected from a shrimp pond on the northern coast of Vietnam were established and morphologically identified as Alexandrium minutum. Nucleotide sequences of domains 1 and 2 of the large subunit ribosomal (LSU) rRNA gene showed high sequence similarity to A. minutum isolates from Malaysia. Paralytic shellfish toxin profile of the clones was characterized by the dominance of GTX4, GTX1, and NEO. GTX3, GTX2, and dcSTX were also present in trace amount. Toxin content varied among the strains and growth stages, ranged from 3.0 to 12.5 fmol cell⁻¹. In addition to these known toxin components, a new gonyautoxin derivative was detected by HPLC, eluting between GTX4 and GTX1. The peak of this compound disappeared under non-oxidizing HPLC condition but unchanged either after treated with 0.05 M ammonium phosphate/10% mercaptoethanol or 0.1N HCl hydrolysis. LCMS ion scanning showed a parental ion of [M + H]⁺ at m/z 396, [M − SO₃]⁺ at m/z 316, and [M − SO₄]⁺ at m/z 298. Based on these results, the derivative was identified as deoxy-GTX4-12ol, and this represents the first report of this toxin analogue.     

Liquid chromatography–electrospray ionization mass spectrometric analysis of corticosterone in rat plasma using selected ion monitoring

A simple and fast yet highly sensitive and specific method based on HPLC coupled to electrospray ionization mass spectrometry has been developed for the quantitation of corticosterone in rat plasma. After extraction of rat plasma (100 μl) with diethyl ether using 5-pregnen-3β-ol-20-one-16α-carbonitrile (Sigma) as internal standard, HPLC was performed on a short C₈ column (Zorbax-Eclipse, 50×4.6 mm I.D.) using a steep methanol–water gradient (methanol 54% to 90% in 6 min). Detection was performed on a single quadruple mass spectrometer in selected ion monitoring mode (m/z 369 for corticosterone and 364 for the internal standard). The detection limit of the assay was 9 fmol (3 pg) of corticosterone on column. In vitro data were subjected to curve fitting (cubic, r²=0.9999). Recovery of corticosterone after extraction ranged from 81 to 93%. The relative standard deviations for intra- and inter-assay precision ranged from 0.8 to 3.6% and 5.2 to 12.9%, respectively. Corticosterone did not undergo any appreciable degradation when stored in plasma at −20°C for 2 months. The assay is routinely used in our laboratory to examine corticosterone levels as a marker of stress in rats and may also be used for the determination of 18-hydroxy-11-deoxycorticosterone.     

Determination of sodium artesunate in plasma using ion-pairing high-performance liquid chromatography

A chromatographic method is described for the determination of sodium artesunate in plasma. This includes cetyltrimethylammonium bromide as a cationic pairing ion in a reversed-phase system using an octadecylsilica 100×4.6 mm I.D. 3 μm analytical column with a mobile phase of acetonitrile/acetate buffer at pH7. Column switching incorporating a 5 μm octadecylsilica 100×4.6 mm I.D. precolumn is used in addition to off-line solid-phase extraction for pretreatment of plasma samples in order to eliminate interference from endogenous components. Detection is by post-column derivatisation with 1.0 M methanolic KOH followed by UV detection at 289 nm. Calibration is linear over the range 100–1600 ng ml⁻¹ and the limit of detection is estimated as 20 ng ml⁻¹. Illustrative results are shown of the artesunate plasma levels determined by the proposed method following the administration of artesunate as tablets and as suppositories to healthy volunteers.     

Simultaneous determination of four anthracyclines and three metabolites in human serum by liquid chromatography–electrospray mass spectrometry

A sensitive and very specific method, using liquid chromatography–electrospray mass spectrometry (LC–ES-MS), was developed for the determination of epirubicin, doxorubicin, daunorubicin, idarubicin and the respective active metabolites of the last three, namely doxorubicinol, daunorubicinol and idarubicinol in human serum, using aclarubicin as internal standard. Once thawed, 0.5-ml serum samples underwent an automated solid-phase extraction, using C₁₈ Bond Elut cartridges (Varian) and a Zymark Rapid-Trace robot. After elution of the compounds with chloroform–2-propanol (4:1, v/v) and evaporation, the residue was reconstituted with a mixture of 5 mM ammonium formate buffer (pH 4.5)–acetonitrile (60:40, v/v). The chromatographic separation was performed using a Symmetry C₁₈, 3.5 μm (150×1 mm I.D.) reversed-phase column, and a mixture of 5 mM ammonium formate buffer (pH 3)–acetonitrile (70:30, v/v) as mobile phase, delivered at 50 μl/min. The compounds were detected in the selected ion monitoring mode using, as quantitation ions, m/z 291 for idarubicin and idarubicinol, m/z 321 for daunorubicin and daunorubicinol, m/z 361 for epirubicin and doxorubicin, m/z 363 for doxorubicinol and m/z 812 for aclarubicin (I.S.). Extraction recovery was between 71 and 105% depending on compounds and concentration. The limit of detection was 0.5 ng/ml for daunorubicin and idarubicinol, 1 ng/ml for doxorubicin, epirubicin and idarubicin, 2 ng/ml for daunorubicinol and 2.5 ng/ml for doxorubicinol. The limit of quantitation (LOQ) was 2.5 ng/ml for doxorubicin, epirubicin and daunorubicinol, and 5 ng/ml for daunorubicin, idarubicin, doxorubicinol and idarubicinol. Linearity was verified from these LOQs up to 2000 ng/ml for the parent drugs (r≥0.992) and 200 ng/ml for the active metabolites (r≥0.985). Above LOQ, the within-day and between-day precision relative standard deviation values were all less than 15%. This assay was applied successfully to the analysis of human serum samples collected in patients administered doxorubicin or daunorubicin intravenously. This method is rapid, reliable, allows an easy sample preparation owing to the automated extraction and a high selectivity owing to MS detection.     

High-performance liquid chromatographic method for determination of DDT and its degradation products in rat plasma, liver and brain: validation and application to a pharmacokinetic study

A sensitive and reliable high-performance liquid chromatographic (HPLC) method, using a solid-phase extraction (SPE), was established and validated for determination of p,p′-DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] and its metabolite p,p′-DDE [1,1-(2,2-dichloroethanylidene)-bis(4-chlorobenzene)] in rat plasma, liver and brain. After being diluted with water, plasma, liver and brain samples were applied to a solid-phase extraction C₁₈ cartridge. The extraction containing p,p′-DDT and p,p′-DDE from the cartridge were cleaned-up using a Florisil Sep-Pak cartridge. The samples were analyzed by HPLC using UV detection at 238 nm. The limit of detection for p,p′-DDT and p,p′-DDE was 0.1 mg kg⁻¹ liver or brain and 0.1 mg l⁻¹ plasma. For six replicate samples at 40, 4 and 0.2 mg kg⁻¹, intra-day precision values were within 4.9% for plasma, 6.4% for liver, and 9.7% for brain. Inter-day precision values at 4 mg kg⁻¹ were within 8.2% for plasma and tissues. The method performances were shown to be selective for p,p′-DDT and p,p′-DDE, and linear over the range 0.04–12 mg kg⁻¹ (mg l⁻¹ for plasma). The absolute recoveries of p,p′-DDT and p,p′-DDE in rat plasma and tissues were over 92%. The method was proved to be applicable to the pharmacokinetic study of DDT in rats after a single oral administration.     

High-performance liquid chromatographic procedure for the quantitative determination of paclitaxel (Taxol) in human plasma

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol^®), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 μm column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10–1000 ng/ml (0.012–1.17 μM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.     

Identification and quantification of five macrolide antibiotics in several tissues, eggs and milk by liquid chromatography–electrospray tandem mass spectrometry

We present an electrospray high-performance liquid chromatographic tandem mass spectrometric (HPLC–MS–MS) method capable of determining in several tissues (muscle, kidney, liver), eggs and milk the following five macrolides: tylosin, tilmicosin, spiramycin, josamycin, erythromycin. Roxithromycin was used as an internal standard. The method uses extraction in a Tris buffer at pH 10.5, followed by protein precipitation with sodium tungstate and clean-up on an Oasis solid-phase extraction column. The HPLC separation was performed on a Purospher C₁₈ column (125×3 mm I.D.) protected by a guard column, with a gradient of aqueous 0.1 M ammonium acetate–acetonitrile as the mobile phase at a flow-rate of 0.7 ml min⁻¹. Protonated molecules served as precursor ions for electrospray ionisation in the positive ion mode and four product ions were chosen for each analyte for multiple reaction monitoring (MRM). A validation study was conducted to confirm the five macrolides by MRM HPLC–MS–MS analysis of a negative control and fortified samples. All of the samples analysed were confirmed with four ions. The ion ratio reproducibility limit ranged from 2.4 to 15%. All compounds could be detected and quantified at half-maximum residue limits (MRLs). The method is specific, quantitative and reproducible enough to conform to European Union recommendations within the concentration range 0.5 MRL–2 MRL (accuracy: 80 to 110%, relative standard deviation: 2 to 13%). This whole method allows extraction and analysis of up to 50 samples per day.     

Sensitive method for the determination of bisphenol-A in serum using two systems of high-performance liquid chromatography

The aim of this study was to establish an easy and accurate method for the determination of bisphenol-A (BPA) in the body liquid such as serum and urine. Two high-performance liquid chromatography (HPLC) systems, HPLC with electrochemical detector (ED), and HPLC with mass spectrometry (MS) using electrospray ionization (ESI) interface were used for the assay in the serum samples prepared with solid-phase extraction method. Water or EtOH at a concentration below 50% was suitable for the extraction of BPA from serum. The limit of detection of BPA was 0.2 ng ml⁻¹ for the HPLC-ED method and 0.1 ng ml⁻¹ for HPLC–MS. There was a good correlation between the data obtained by the two HPLC systems. BPA concentrations in healthy human serum were low (0–1.6 ng ml⁻¹). From various commercial fetal bovine serum and sheep plasma, however, significant amounts of BPA were detected. Since no BPA was detected from sheep plasma immediately after collection, the high amounts of BPA were considered to be caused by the handling of blood during the preparation of the products after blood collection. In vitro study showed that the amount of BPA leached from polycarbonate tube into sheep plasma were 40 times larger than those into water and the leached amount of BPA depended on the temperature (37°C>20°C>5°C).     

Determination of naphthodianthrones in plant extracts from Hypericum perforatum L. by liquid chromatography–electrospray mass spectrometry

The determination of the naphthodianthrone constituents in extracts of dried blossoms of Hypericum perforatum L. by combined HPLC–electrospray mass spectrometry is described. Hypericin (1), pseudohypericin (2) and their precursor compounds produce intensive negative quasi-molecular ions by deprotonation provided a non-acidic eluent system is used in the HPLC separation. From the [M–H]⁻ ions formed in the electrospray ionization process characteristic daughter ion spectra can be obtained by collisional activation which have been studied by tandem mass spectrometry.     

Determination of gacyclidine enantiomers in human plasma by gas chromatography–mass spectrometry using selected-ion monitoring

A sensitive gas chromatographic assay using mass selective-detection has been developed for the simultaneous quantitation of the enantiomers of (±)-gacyclidine (a non competitive N-methyl-

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-aspartate antagonist) in human plasma. Gacyclidine enantiomers and phencyclidine (PCP), the internal standard, were extracted using a single-step liquid–liquid extraction with hexane at pH 8.0. Each enantiomer was separated on a chiral gas chromatography capillary column and specifically detected by mass spectrometry (MS) in selected-ion monitoring (SIM) mode. Gacyclidine enantiomers and PCP were monitored using the fragment ions at m/z 206 and 200, respectively. No interference was observed from endogenous components. The limit of quantitation (LOQ) for each enantiomer of gacyclidine was 300 pg/ml by using plasma samples of 500 μl. The calibration curves were linear (r²=0.998) over a range of 0.3125 to 20 ng/ml. The extraction efficiency was higher than 95% for both enantiomers. Intra- and inter-day bias were less than 10% at every standard curve concentration. Intra-day precision was less than 19% for (−)-gacyclidine and 15% for (+)-gacyclidine. Inter-day precision was below 15% for both enantiomers. The assay was validated for an enantioselective pharmacokinetic study in healthy male volunteers.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

Determination of a HIV protease inhibitor (DMP 450) in animal and human plasma by solid-phase extraction and high-performance liquid chromatography

Extraction of DMP 450 from plasma was performed with C₂ solid-phase extraction columns, using 0.1 M ammonium acetate in 90% methanol to elute DMP 450. The extraction recovery over the range of 10 to 10 000 ng/ml averaged 81.0, 96.2, 77.4, 95.2 and 68.0% from rat, dog, monkey, chimpanzee (25–10 000 ng/ml) and human plasma, respectively. HPLC analysis was carried out with a C₁₈ column and a mobile phase of acetonitrile, methanol and 30 mM potassium phosphate (pH 3), the composition dependent on the type of plasma being analyzed, and monitored at a wavelength of 229 nm. Intra-day and inter-day coefficients of variation were less than 9.9 and 12.9%, respectively. Absolute differences were less than 11.5%.     

Determination of

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 Å), 150×4.6 mm C₈ analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor→product ion combinations of m/z 1093.7→1033.6 and 1094.7→1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–1000 ng/ml using ISP, and 2.5–500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.     

Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 Å), 150×4.6 mm C₈ analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor→product ion combinations of m/z 1093.7→1033.6 and 1094.7→1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–1000 ng/ml using ISP, and 2.5–500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.     

Determination of the GABAB receptor agonist CGP 44532 (3-amino-2-hydroxypropylmethylphosphinic acid) in rat plasma after pre-column derivatization by micro-high-performance liquid chromatography combined with negative electrospray tandem mass spectrometry

An assay, based on pre-column derivatization and micro-high-performance liquid chromatography–tandem mass spectrometry, was developed for the determination of the GABA_B agonist CGP 44532 in rat plasma. CGP 44532, a highly polar 3-amino-2(S)-hydroxypropylmethylphosphinic acid, presented difficulties in developing a chromatographic method for the analysis of the compound in rat plasma. Instead of analyzing the target compound directly, it was derivatized prior to separation to a 4-nitrobenzylcarbamate isopropyliden derivative. In order to reach the required quantitation limit, on-line solid-phase extraction was utilized for sample clean-up and reversed-phase micro-column high-performance liquid chromatography, for separation of the plasma samples. The separated compounds were detected by negative electrospray tandem mass spectrometry in selected reaction monitoring mode. The derivatives show good chromatographic and mass spectrometric properties and both the target compound and the internal standard, could be eluted as symmetrical peaks with good signal/noise ratio. The MS–MS detection was selective and sensitive due to the straight fragmentation pattern. After injection of 200-μl sample aliquots, the limit of quantification was 10 ng ml⁻¹. The analytical assay is useable in the range of 10–500 ng ml⁻¹.     

Sulfalene concentrations in plasma and blood cells of Plasmodium falciparum malaria cases after treatment with metakelfin using high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method using acetonitrile–methanol–1 M perchloric acid–water (25:9:0.8:95, v/v/v) at a flow-rate of 1.0 ml min⁻¹ on LiChrospher 100 RP 18 column (250×4 mm; 5 μm) with UV (254 nm) detection has been developed for the determination of sulfalene in plasma and blood cells after oral administration of the antimalarial drug metakelfin. Calibration curves were linear in the range 0.5–100 μg ml⁻¹. The limit of quantification was 50 ng ml⁻¹. Within-day and day-to-day coefficients of variation averaged 3.84 and 5.31%, respectively. Mean extraction recoveries of sulfalene from plasma and blood cells were 87.21 and 84.65%, respectively. Mean concentrations of sulfalene in plasma of P. falciparum cases on days 2, 7 and 15 were 44.58, 14.90 and 1.70 μg ml⁻¹, respectively; in blood cells concentrations of sulfalene were 7.77, 3.25 and 0.75 μg ml⁻¹, respectively, after oral treatment with two tablets (1000 mg) of metakelfin. Significant difference was recorded on day 2 for sulfalene concentration in blood cells of healthy and P. falciparum cases (t=9.49; P<0.001).