Determination of fluorescent trypanocidal diamidines by quantitative thin-layer chromatography

The fluorescent trypanocidal diamidines 2-(4-amidinophenyl)indole-6-carboxamidine dihydrochloride (I, DAPI), 2-(4-amidinophenyl)benzo[b]thiophene-6-carboxamidine dihydrochloride (II) and 2-(4-amidinophenyl)-1-benzofurane-5-carboxamidine dihydrochloride (III) were determined in plasma, urine, faeces and tissues of experimental animals using quantitative thin-layer chromatography. Samples were extracted with n-octanol after addition of sodium hydroxide and subsequently re-extracted into 0.1 M hydrochloric acid. Chromatography was performed on silica gel plates under nitrogen with n-butanol saturated with 2 M hydrochloric acid. Quantitation was performed by measuring native fluorescence using a fluorodensitometer. The respective diamidines were used as internal standards for each other to ensure precision (coefficient of variation less than 7%) and accuracy of the assay. Calibration curves were linear up to 150 ng/ml of sample solution with detection limits of 10 ng/ml of sample solution for I and III and 50 ng/ml for II. The described method has been successfully used for pharmacokinetic studies in experimental animals.     

Rapid, high-sensitivity reversed-phase liquid chromatographic assay for 9-chloro-2-(2-furyl) [1,2,4]triazolo[1,5-c]quinazolin-5-imine and its oxo metabolite in plasma using fluorescence detection

A rapid, sensitive and specific assay for 9-chloro-2-(2-furyl) [1,2,4]triazolo[1,5-c]quinazolin-5-imine (I) and its oxo metabolite (II) in plasma was developed and validated employing reversed-phase high-performance liquid chromatography with fluorescence detection. Sample preparation was achieved by a simple ethyl acetate extraction from plasma buffered at pH 10 (0.1 M boric acid-0.1 M potassium chloride). Chromatographic analyses were performed isocratically on a C18 column, with a mobile phase consisting of methanol-0.2 M sodium acetate buffer, pH 5.0 (67:33, v/v). Chromatographic run time was less than 8 min. The assay was linear (r greater than 0.9998) over the concentration range 1.50-10,000 ng/ml for both I and II; for individual studies, curves covering a range of two orders of magnitude were generally employed. Limits of detection for I and II were 0.5 and 1.0 ng/ml, respectively. A preliminary investigation of the plasma concentrations of I and II in the rat following a single 30 mg/kg oral dose demonstrated the applicability of the method for pharmacokinetic studies.     

Automated high-performance liquid chromatographic method for the simultaneous determination of cefotiam and delta 3-cefotiam in human plasma using column switching.

An automated high-performance liquid chromatographic method using column switching was established for the simultaneous determination of cefotiam (I) and delta 3-cefotiam (II) in human plasma after oral administration of cefotiam hexetil dihydrochloride. The method allowed the determination of analytes in plasma by the direct injection of diluted specimen with phosphate buffer. The analytes were enriched onto the C18 short pretreatment column by 0.05 M phosphate buffer (pH 7.7), while proteins and endogenous hydrophilic substances in plasma were washed off to waste. The enriched analytes were then back-flushed onto the analytical C18 column, separated by a mixture of 0.05 M phosphate buffer (pH 7.7)-acetonitrile (88:12, v/v) and detected by the ultraviolet absorbance at 254 nm. Recoveries from spiked plasma were quantitative, and the coefficients of variation were below 4%. The lower detection limits in plasma were 10 ng/ml for both I and II. Concentrations of I and II in plasma determined by the present method were in good agreement with those obtained by the conventional deproteinization method.     

Determination of a new calcium antagonist, sesamodil fumarate (SD-3211), and its metabolite in plasma by liquid chromatography with electrochemical detection

A sensitive and selective high-performance liquid chromatographic method is described for the determination of a novel calcium antagonist, (+)-(R)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-[(3,4- methylenedioxy)phenoxy]ethyl]amino]propoxy]phenyl]-4-methyl-3-oxo-2H- 1,4-benzothiazine hydrogen fumarate (sesamodil fumarate; JAN, SD-3211, I), and its N-desmethylated metabolite (II) in plasma. Compounds I and II and an internal standard were isolated from plasma by solid-phase and liquid-liquid extraction. The extract was chromatographed on a reversed-phase C18 column, and the compounds of interest were detected by dual coulometric electrodes operated in an oxidative screen mode. The limit of determination for both I and II was at least 0.4 ng/ml in plasma. The utility of the assay was demonstrated by determining plasma levels of I and II in five dogs administered an oral dose of 60 mg of the drug.     

Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients.

CPT-11 (I; 7-ethyl-10-[4-(1-piperidino)-1- piperidino]carbonyloxycamptothecin) is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C18) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C18 reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml-10 micrograms/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5-1000 ng/ml) was 13.0% (range 4.9-19.4%) for I and 12.8% (6.7-19.1%) for II; the between-day R.S.D. (5-10,000 ng/ml was 7.9% (5.4-17.5%) for I and 9.7% (3.5-15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m2 I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 +/- 285 ng/ml (mean +/- standard error of the mean). Plasma decay was triphasic with half-lives alpha, beta and gamma of 5.4 +/- 1.8 min, 2.5 +/- 0.5 h and 20.2 +/- 4.6 h, respectively. The volume of distribution at steady state was 105 +/- 15 l/m2, and the total body clearance was 12.5 +/- 1.9 l/h.m2. The maximum concentrations of the active metabolite II reached 36 +/- 11 ng/ml.     

Determination of midazolam and its major hydroxy metabolite in plasma by gas liquid chromatography. Application to a pharmacokinetic study

A sensitive gas liquid chromatographic (GLC) assay was developed for plasma determinations of 8-chloro-6-(2′ -fluorophenyl)-1-methyl-4H-imidazo[1,5 α][1,4]benzodiazepine (compound I) and its hydroxymethylimidazo metabolite (compound II). The internal standards used were 8-chloro-6-(2′ -chlorophenyl)-1 -methyl-4H-imidazo[1,5 α][1,4]benzodiazepine (compound VI) and 7-chloro-5-(2′ -fluorophenyl)-1, 3-dihydro-1-(hydroxyethyl)-2H-1,4-benzodiazepin-2-one (compound VII) for compounds I and II, respectively. Following extraction, and silylation for compound II, compounds I and II were analyzed by GLC using a glass column packed with 5% OV-101 on Gas-Chrom Q, and a ⁶³Ni electron-capture detector. The GLC method was validated by a CI-GC/MS technique. The detection limit of the assay is approximately 4–5 ng/ml for compound I and 3 ng/ml for compound II. The method was used in comparative pharmacokinetic studies of the distribution of the two compounds in arterial and venous blood.     

Determination of the antiallergenic agent, N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H- pyrido [2,1-b]quinazoline-8-carboxamide, in plasma by reversed-phase high-performance liquid chromatographic analysis using fluorometric detection

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[ 2,1-b] quinazoline-8-carboxamide (I), and its major metabolite, 2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxylic acid (I-A), in plasma. The assay involves precipitation of the plasma proteins with acetonitrile--methanol (9:1), followed by the analysis of an aliquot of the protein-free filtrate by reversed-phase ion-pair HPLC with fluorescence detection for quantitation. The analogous compound, N-[6-(1H-imidazol-1-yl)hexyl]-2-(1-methylethyl)-11-oxo-11H-pyrido [2,1-b]-quinazoline-8-carboxamide (II), is used as the internal standard. The overall recovery of compounds I and I-A from plasma is 107.0 +/- 8.6% and 107.0 +/- 10.0%, respectively. The sensitivity limits of quantitation are 20 ng of I, and 10 ng of I-A per ml of plasma using a 0.5-ml aliquot. The assay was used to monitor the plasma concentrations of I and of I-A in a dog following a 5 mg/kg intravenous infusion of I . 2HCl, a 10 mg/kg oral dose of I . 2HCl and of metabolite I-A.     

High-performance liquid chromatographic determination of N-[2(S)-(mercaptomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-methionine, the active plasma metabolite of a prodrug atriopeptidase inhibitor (SCH 42495), using a thiol selective (Au/Hg) amperometric detector.

A high-performance liquid chromatographic assay for the determination of N-[2(S)-(mercaptomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-methionine (SCH 42354; II), the active metabolite of the atriopeptidase inhibitor prodrug, N-[2(S)-(acetylthiomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L-methi onine ethyl ester (SCH 42495; I), in human plasma was validated for use in clinical pharmacokinetic studies. Plasma (200 microliters) was processed by protein precipitation with acetone containing the internal standard, N-[2(S)-(mercaptomethyl)-3-(2-methylphenyl)-1-oxopropyl]-L- ethionine (III). Compound II was recovered (ca. 90%) in the supernatant after centrifugation and prepared for injection by the addition of 0.15 M monochloroacetic acid containing 0.2 mM EDTA. Separation of II and III was accomplished on commercially available reversed-phase C8 columns designed for the separation of basic compounds. Both compounds were detected using amperometric detection (+0.125 V versus Ag/AgCl) on a thin-layer Au/Hg amalgam electrode. The lower limit of quantitation was 10 ng/ml, where the inter-assay precision (coefficient of variation) was +/- 11.4% and the inter-assay accuracy (bias) was +1.0%. No endogenous interferences were observed in the extracts obtained from drug-free plasma. The detector response (using either peak area or height ratios of II to III) was linear from 0.01 to 1.0 micrograms/ml. Compound II was stable in plasma supplemented with EDTA and sodium hydrogensulfite for at least 3 months when stored frozen at -78 degrees C; no significant decomposition of II was observed following three freeze-thaw cycles. The feasibility of this liquid chromatographic assay with electrochemical detection was demonstrated with plasma samples from hypertensive subjects administered 100 mg of compound I.     

Determination of a new non-benzodiazepine anxiolytic and its O-demethyl metabolite in plasma by high-performance liquid chromatography using automated column-switching

An highly sensitive and fully automated high-performance liquid chromatographic assay was developed for the determination of a novel non-benzodiazepine anxiolytic (I) [(R)-2-(methoxymethyl)-1-[(7-oxo-8-phenyl-7H-thieno[2,3-a]quinolizin+ ++- 10-yl)carbonyl]pyrrolidine] and its O-demethyl metabolite (II) in plasma, using column-switching for direct injection of plasma samples. After dilution in internal standard solution, the sample was injected onto a pre-column (17 mm x 4.6 mm) dry-packed with pellicular C18 reversed-phase material. Polar plasma components were removed by flushing the pre-column with water-acetonitrile (90:10, v/v). Retained substances, including I and II, were backflushed onto an analytical column, separated by gradient elution and detected by means of fluorescence detection (excitation, 304 nm; emission, 475 nm). After washing the analytical column and re-equilibrating the pre-column, the system was ready for the next injection. The limit of quantification for I and II was 0.25 and 0.5 ng/ml, respectively, using a 350-microliter specimen of plasma. The practicability of the new method was demonstrated by analysis of more than 300 plasma samples from a tolerance study performed with human volunteers. Owing to its high sensitivity, the method can be used to calculate pharmacokinetic parameters of compounds I and II in man after a single oral dose of about 1 mg of I.     

High-performance liquid chromatographic assay of alpha-(4-fluorophenyl)-4-(5-fluoro-2-pyrimidinyl)-1-piperazine butanol (BMY 14802), a potential antipsychotic drug, in monkey and rat plasma

A high-performance liquid chromatographic-fluorescence method was developed for the quantitative analysis of BMY-14802 (I) in monkey and rat plasma. After the addition of the internal standard (BMY-14853 I.S.), 250 microliters of plasma were made basic by the addition of 2 ml of saturated sodium carbonate buffer. Compound I and the I.S. were then extracted into 5 ml of methyl tert.-butyl ether. The organic phase was evaporated and the resulting residue was reconstituted in mobile phase. Final separation and quantitation of I was achieved on an octadecyl column with a 0.05 M potassium phosphate-acetonitrile-triethylamine-85% phosphoric acid (650:350:0.1:0.05, v/v) mobile phase. Fluorescence detection was used to monitor the eluent at an excitation wavelength of 240 nm and an emission wavelength of 400 nm. The limit of detection was 0.5 ng/ml. The standard curve was linear over the range 5.0-1000 ng/ml. Intra-assay and inter-assay precision values were less than 4.0% relative standard deviation and accuracy was within 12% of nominal values. Compound I was shown to be stable in monkey and rat plasma for at least six months when stored at -20 degrees C.     

Determination of the anxiolytic agent 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide in whole blood, plasma or urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-alpha]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-alpha] [1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether-methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 +/- 5.4% (S.D.) and 90.3 +/- 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration-time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Simultaneous determination of vinburnine and 6-hydroxyvinburnine in human plasma by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method has been developed to allow the simple and rapid determination of both vinburnine (I) and its main metabolite, 6-hydroxyvinburnine (II), in heparinized human plasma (0.5 ml). Compounds I and II and p-chlorodisopyramide (internal standard) were first extracted with alkalinized ethyl acetate and then with sulphuric acid. Separation was achieved on a reversed-phase muBondapak C18 column with a mobile phase of acetonitrile-water-0.1 M heptanesulphonate in acetic acid and with detection at 254 nm. Each run required 20 min. The within-day coefficients of variation for identical samples (20 ng/ml) were 7 and 6% and between-day coefficients of variation 8 and 26% for I and II, respectively. The detection limit was 5 ng/ml (normal therapeutic concentration, 10-300 ng/ml). The application of the method to drug monitoring was compared to that of a thin-layer chromatographic procedure.     

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.     

Reversed-phase ion-pair partition liquid chromatography of chelates with 2-(3,5-dibromo-2-pyridylazo)-5-[N-ethyl-N-(3-sulphopropyl) amino] phenol and analogues

The ion-pair reversed-phase chromatography of some transition metal chelates with 2-(3,5-dibromo-2-pyridylazo)-5-[N-ethyl-N-(3-sulphopropyl)amino]phenol (3,5-diBr-PAESPAP) was studied. 3,5-DiBr-PAESPAP and its V(V), Cr(III), Fe(II), Co(III) and Ni(II) chelates were retained on and the copper (II), zinc(II) and cadmium(II) chelates dissociated in an ODS column using acetonitrile/water (37+63, v/v) (pH 7.0) containing 0.01 M acetate, 0.01 M 3-(N-morpholino)propanesulphonate buffer (pH 7.0) and 0.05 M Na⁺ as mobile phase. The chromatograms of 3,5-diBr-PAESPAP chelates were compared with those of the chelates with 2-(3,5-dibromo- 2-pyridylazo)-5-[N-(3-sulphopropyl)amino]phenol (3,5-diBr-PASPAP),2-(5-bromo-2- pyridylazo)-5-[N-(3-sulphopropyl)amino]phenol and 2-(5-bromo-2-pyridylazo)-5-[N-propyl-N- (3-sulphopropyl)amino] phenol. With 3,5-diBr-PAESPAP the Fe(II) and Ni(II) chelates were not resolved, but resolution was achieved with 3,5-diBr-PASPAP. The calibration graphs were linear over the ranges 2.0–10.0 ng (10-μl injection) of Fe, Ni and Co and for 20–100 ng (10-μl injection) for V with 3,5-diBr-PAESPAP and 3,5-diBr-PASPAP.     

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by liquid chromatography electrospray ionization mass spectrometry and its application to pharmacokinetic studies

A simple and sensitive method was developed for the simultaneous quantification of harpagoside and cinnamic acid in rat plasma using high-performance liquid chromatography system coupled to a negative ion electrospray mass spectrometric analysis. The plasma sample preparation was a simple deproteinization by the addition of two volumes of acetonitrile. The analytes were separated on an Intersil C8-3 column (2.1 mm i.d.x250 mm, 5 microm) with acetonitrile-5 mm ammonium formate aqueous solution (60:40, v/v) as mobile phase at a flow-rate of 0.2 mL/min. Detection was performed on a quadrupole mass spectrometer equipped with electrospray ionization (ESI) source operated under selected ion monitoring (SIM) mode. [M+HCOO]- at m/z 539 for harpagoside, [M-H]- at m/z 147 for cinnamic acid and [M-H]- at m/z 137 for salylic acid (internal standard) were selected as detecting ions, respectively. The method was validated over the concentration range 7-250 ng/mL for harpagoside and 5-500 ng/mL for cinnamic acid. The lower limits of quantitation for harpagoside and cinnamic acid were 7 and 5 ng/mL, respectively. The intra- and inter-day precisions (RSD%) were within 9.5% and the assay accuracies (RE%) ranged from -5.3 to 3.0% for both analytes. Their average recoveries were greater than 86%. Both analytes were proved to be stable during all sample storage, preparation and analysis procedures. The method was successfully applied to the pharmacokinetic study of harpagoside and cinnamic acid following oral administration of Radix Scrophulariae extract to rats.     

Simultaneous determination of paclitaxel and a new P-glycoprotein inhibitor HM-30181 in rat plasma by liquid chromatography with tandem mass spectrometry

An LC-MS/MS method for the simultaneous determination of a new P-glycoprotein inhibitor 4-oxo-4H-chromene-2-carboxylic acid [2-(2-(4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-phenyl)-2H-tetrazol-5-yl)-4,5-dimethoxy-phenyl]-amide (HM-30181) and a P-glycoprotein substrate paclitaxel in rat plasma was developed to simultaneously evaluate the pharmacokinetics of paclitaxel and HM-30181 in the rats. HM-30181, paclitaxel, HM-30059 (internal standard (I.S.) for HM-30181), and docetaxel (I.S. for paclitaxel) were extracted from rat plasma with methyl-tert-butyl ether and analyzed on an Atlantis C18 column (5 microm, 2.1 x 100 mm) with the mobile phase of ACN/10 mM ammonium formate (75:25 v/v). The analytes were detected using an ESI MS/MS in the multiple reaction monitoring (MRM) mode. The standard curves for HM-30181 and paclitaxel in plasma were linear (r > 0.999) over the concentration range of 2.0-500 ng/mL with a weighting of 1/concentration2. The method showed a satisfactory sensitivity (2 ng/mL using 50 microL plasma), precision (CV: < or = 6.6%), accuracy (relative error: -6.3 to 2.0%), and selectivity. This method was successfully applied to the pharmacokinetic study of HM-30181 and paclitaxel in rat plasma after oral-coadministration of paclitaxel and HM-30181 to male Sprague- Dawley rats.     

Determination of dextromethorphan and metabolites in human plasma and urine by high-performance liquid chromatography with fluorescence detection

Sensitive methods were developed for the analysis of dextromethorphan (I) and two metabolites, (+)-17-methyl-morphinan-3-ol (II) and (+)-morphinan-3-ol (III), in plasma as well as dextromethorphan and three metabolites II, III and (+)-3-methoxymorphinan (IV) in urine using high-performance liquid chromatography followed by detection with a fluorometer. Dextromethorphan and its metabolites were extracted from plasma and urine and separated in the reversed-phase mode. The practical lower limits of determination for I, II, and III in plasma were 0.5, 5, and 5 ng/ml, respectively; for I, II, III, and IV in urine, the limits were 20 ng/ml, 0.6 microgram/ml, 0.5 microgram/ml, and 15 ng/ml, respectively. The linearity of the calibration graphs was excellent (r varied from 0.9994 to 0.9999) over concentration ranges of two orders of magnitude.     

Determination of the new anticancer agent KW 2149, 7-N-[2-[2-(gamma-L-glutamylamino)ethyl)dithio)ethyl]mitomycin C, an analogue of mitomycin C.

The new mitomycin 7-N-[2-[2-(gamma-L-glutamylamino)ethyl)dithio)ethyl] mitomycin C (KW 2149) (I) proved to be active against a wide variety of experimental tumours. In order to perform pharmacokinetic studies with the new drug in Phase I sessions, a fast and reliable method has been developed based on the data of previous assays for mitomycin C. XAD-2 was preferred for isolation of I from blood plasma. The recovery of I was 50% whereas that of mitomycin C was 85%. Optimal separation was obtained on octadecyl silica columns with methanol-water (45:55, v/v) as mobile phase, while ultraviolet absorbance detection was performed at 375 nm. The assay enabled determination of I in a plasma concentration range of 20-1000 ng/ml using porfiromycin as internal standard.     

Quantitative determination of marine toxins associated with diarrhetic shellfish poisoning by liquid chromatography coupled with mass spectrometry

Quantitative determination by liquid chromatography (LC) coupled with mass spectrometry (MS) was achieved for the following 10 toxins found in association with diarrhetic shellfish poisoning: okadaic acid (OA), dinophysistoxin-1 (DTX1), 7-O-palmitoylokadaic acid (palOA), 7-O-palmitoyldinophysistoxin-1 (pa1DTX1), pectenotoxin-1 (PTX1), pectenotoxin-2 (PTX2), pectenotoxin-2 seco acid (PTX2SA), pectenotoxin-6 (PTX6), yessotoxin (YTX), and 45-hydroxyyessotoxin (YTXOH). Toxins in 2 g of the adductor muscle or the digestive glands of scallops, Patinopecten yessoensis, were extracted with 18 ml of methanol-water (9:1, v/v), freed of polar contaminants by partition between chloroform and water, and treated by solid-phase extraction on a silica cartridge column. Samples containing YTXOH were purified separately on a buffered reversed-phase column. Chromatographic separation was achieved by the following combinations of columns and mobile phases: a Symmetry C18 column with acetonitrile-0.05% acetic acid (7:3, v/v) for OA, DTX1, PTX6 and PTX2SA; a Develosil ODS column with the same mobile phase for PTX1 and PTX2; a Capcellpak column with methanol-2.5% acetic acid (98:2, v/v) for palOA and palDTX1; and an Inertsil ODS column with methanol-0.2 M ammonium acetate (8:2, v/v) for YTX and YTXOH. Carboxylic acid toxins were selectively monitored on [M-H]- ions, sulfated toxins on [M-Na]-ions, and neutral toxins on [M+NH4]+ ions. Average recoveries of the toxins spiked to tissue homogenates ranged from 70 to 134%. Detection limits in the muscle ranged from 5 to 40 ng/g and those in the digestive glands from 10 to 80 ng/g.     

Sensitive determination of piritramide in human plasma by gas chromatography.

A selective and sensitive method for the determination of piritramide in human plasma is described. A 1-ml aliquot of plasma was extracted with 10 ml of hexane-isoamyl alcohol (99.5:0.5, v/v) (extraction efficiency 86%) after addition of 50 microliters of 2 M ammonia and 20 microliters of aqueous strychnine solution (100 ng per 10 microliters) as internal standard. Gas chromatography was performed with J&W DB-1, 30 m x 0.53 mm I.D. separation column, film thickness 1.5 microns, using an nitrogen-phosphorus-sensitive detector. The assay was linear in the concentration range 3.75-2250 ng/ml (r = 0.999), with a lower limit of detection of 1-2 ng/ml. The precision was determined using spiked plasma samples (10 and 50 ng/ml), with coefficients of variation of 3.5 and 3.1% (intra-day; n = 5) and 4.6 and 4.1% (inter-day; n = 4). In the range 3.75-150 ng/ml, the accuracy of the assay was 3.36%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or post-operative analgesia.