Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC-MS-MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC-MS-MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC-MS interface. The standard curve range in plasma was 0.5-50 ng/ml for MOR, 1.0-100 ng/ml for M6G, and 10-1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC-MS-MS systems to analyze over one thousand samples from clinical trials.     

Simple and rapid determination of irinotecan and its metabolite SN-38 in plasma by high-performance liquid-chromatography: application to clinical pharmacokinetic studies

Irinotecan (CPT-11) is an anticancer agent widely employed in the treatment of colorectal carcinoma. A simple, rapid and sensitive high-performance liquid chromatographic method for the simultaneous determination of CPT-11 and its metabolite SN-38 in plasma, and their preliminary clinical pharmacokinetics are described. Both deproteinisation of plasma specimens (100 microl) and addition of the internal standard, camptothecin (CPT), are achieved by incorporating to samples 100 microl of a solution of CPT (1 microg/ml) in acetonitrile-1 mM orthophosphoric acid (90:10); 200 microl of this acidified acetonitrile solution, drug-free, is also added to accomplish complete deproteinisation: this procedure reduces sample preparation time to a minimum. After deproteinisation, samples are treated with potassium dihydrogenphosphate (0.1 M) and injected into a Nucleosil C18 (5 microm, 250 x 4.0 mm) column. Mobile phase consists of potassium dihydrogenphosphate (0.1 M)-acetonitrile (67:33), at a flow-rate of 1 ml/min. CPT-11, SN-38 and CPT are detected by fluorescence with excitation wavelength set at 228 nm and emission wavelengths of CPT-11, SN-38 and CPT fixed, respectively, at 450, 543 and 433 nm. The limits of quantitation for CPT-11 and SN-38 are 1.0 and 0.5 ng/ml, respectively. This method shows good precision: the within day relative standard deviation (RSD) for CPT-11 (1-10000 ng/ml) is 5.17% (range 2.15-8.27%) and for SN-38 (0.5-400 ng/ml) is 4.33% (1.32-7.78%); the between-day RSDs for CPT-11 and SN-38, in the previously described ranges, are 6.82% (5.03-10.8%) and 4.94% (2.09-9.30%), respectively. Using this assay, plasma pharmacokinetics of CPT-11, SN-38 and its glucuronidated form, SN-38G, have been determined in one patient receiving 200 mg/m2 of CPT-11 as a 90 min intravenous infusion. The peak plasma concentration of CPT-11 at the end of the infusion is 3800 ng/ml. Plasma decay is biphasic with a terminal half-life of 11.6 h. The volume of distribution at steady state (Vss) is 203 l/m2, and the total body clearance (Cl) is 14.8 l/h x m2. The maximum concentrations of SN-38 and SN-38G reach 28.9 and 151 ng/ml, respectively.     

Determination of non-protein bound phenylbutazone in bovine plasma using ultrafiltration and liquid chromatography with ultraviolet detection

A liquid chromatographic procedure using UV detection was coupled with ultrafiltration for the quantitation of free phenylbutazone in bovine plasma, in the range of 20 ng/ml to 2.0 microg/ml. Whole plasma samples (0.5 to 1 ml) were placed in a 2-ml centrifugal concentrator with a molecular-mass cut-off membrane of 10000 and centrifuged at 4500 g for 2 h at 4 degrees C using a fixed angle rotor. The ultrafiltrate was transferred to an LC vial with a 200-microl insert and 100 microl was injected into an LC system. The chromatographic system used a C18 reversed-phase column connected to a UV detector set at 264 nm. The mobile phase was 0.2 M sodium phosphate buffer (pH 7)-methanol (1:1). Recoveries of phenylbutazone from protein-free plasma water fortified at levels of 20 ng/ml to 2 microg/ml ranged from 91 to 93%, with relative standard deviations (R.S.D.s) ranging from 1 to 4%. The concentration of incurred non-protein bound phenylbutazone obtained from a cow intravenously dosed twice with 2 g phenylbutazone, 8 h apart, was 111, 26 and 11 ng/ml for 2, 72 and 104 h post first phenylbutazone dose, respectively.     

Mycoplasma hyopneumoniae increases intracellular calcium release in porcine ciliated tracheal cells

We investigated the effects of intact pathogenic Mycoplasma hyopneumoniae, nonpathogenic M. hyopneumoniae, and Mycoplasma flocculare on intracellular free Ca2+ concentrations ([Ca2+]i) in porcine ciliated tracheal epithelial cells. The ciliated epithelial cells had basal [Ca2+]i of 103 +/- 3 nM (n = 217 cells). The [Ca2+]i increased by 250 +/- 19 nM (n = 47 cells) from the basal level within 100 s of the addition of pathogenic M. hyopneumoniae strain 91-3 (300 microg/ml), and this increase lasted approximately 60 s. In contrast, nonpathogenic M. hyopneumoniae and M. flocculare at concentrations of 300 microg/ml failed to increase [Ca2+]i. In Ca2+-free medium, pathogenic M. hyopneumoniae still increased [Ca2+]i in tracheal cells. Pretreatment with thapsigargin (1 microM for 30 min), which depleted the Ca2+ store in the endoplasmic reticulum, abolished the effect of M. hyoneumoniae. Pretreatment with pertussis toxin (100 ng/ml for 3 h) or U-73122 (2 microM for 100 s), an inhibitor of phospholipase C, also abolished the effect of M. hyopneumoniae. The administration of mastoparan 7, an activator of pertussis toxin-sensitive proteins G(i) and G(o), increased [Ca2+]i in ciliated tracheal cells. These results suggest that pathogenic M. hyopneumoniae activates receptors that are coupled to G(i) or G(o), which in turn activates a phospholipase C pathway, thereby releasing Ca2+ from the endoplasmic reticulum. Thus, an increase in Ca2+ may serve as a signal for the pathogenesis of M. hyopneumoniae.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether-isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 microl potassium phosphate (0.1 M, pH 2.2) and 60 microl of the acid solution was injected into a C18 BDS Hypersil analytical column (3 microm, 100x4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H3PO4)-acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5-100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

Factors influencing the protein binding of IQO4, a new isoquinolinedione derivative

Various factors most likely to influence the plasma protein binding of IQO4, a new isoquinolinedione derivative, to 4% human serum albumin (HSA) were evaluated using an equilibrium dialysis technique at an initial IQO4 concentration of 5 microg/ml. It took approximately 12 h incubation to reach an equilibrium between 4% HSA and isotonic S?rensen phosphate buffer at pH 7.4 containing 3% dextran ('the buffer') using a Spectra/Por 2 membrane (molecular weight cut-off, 12000-14000) in a water-bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. IQO4 was stable both in 4% HSA and in 'the buffer' for up to 24 h incubation at 37 degrees C. The binding of IQO4 was constant (89.9 +/- 1.40%, mean +/- standard deviation) at IQO4 concentrations ranging from 1 to 100 microg/ml. However, the extent of binding was dependent on HSA concentrations. The values were 32.5, 62.0, 79.1, 84.9, 90.9, 91.2, and 91.7% at HSA concentrations of 0.5, 1, 2, 3, 4, 5, and 6%, respectively; on incubation temperature, 96.7, 93.8, and 91.0% when incubated at 4, 22, and 37 degrees C, respectively; and on the buffer pHs, 84.4, 87.2, 88.2, 90.9, and 92.3% for the buffer pHs of 5.8, 6.4, 7, 7.4, and 8, respectively. The free fraction of IQO4 increased with the addition of sulfisoxazole (0-300 microg/ml), and salicylic acid (0-300 microg/ml). The protein binding of IQO4 was independent of the quantity of alpha-1-acid glycoprotein (up to 0.32%), chloride ion (up to 0.546%) and heparin (up to 40 units/ml).     

Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, phenylbutazone and oxyphenbutazone in equine serum by high-performance liquid chromatography

Ethyl acetate extracts of equine serum, containing 0-5 microg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile-water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 microg/ml for HYD, DEX and IND, 0.5 microg/ml for PB and 1 microg/ml for OPB, despite the occurrence of significant degradation of OPB and PB during the analysis) and precision (intra-assay and inter-assay C.V.'s of about 3-6 and 9-15%, respectively) of the method appeared appropriate for anti-doping control of racehorses.     

Protective effect of ketoprofen lysine salt on interleukin-1β and bradykinin induced inflammatory changes in hamster cheek pouch microcirculation

OBJECTIVE: The purpose of this study was to assess the efficacy of topically applied ketoprofen lysine salt (KLS), a cyclooxygenase inhibitor, against the inflammatory changes induced by interleukin-1beta (IL-1beta) and bradykinin (BK) in hamster cheek pouch microcirculation. In addition, we characterised the pharmacological regulation of IL-1beta activity in this model. MATERIALS AND METHODS: Male Syrian hamsters were used. Microcirculation was visualised by fluorescent microscopy. Leukocyte adhesion, permeability, perfused capillary length (PCL) and capillary red blood cell (RBC) velocity were evaluated. TREATMENTS: KLS (25 microg/ml/min to 1.6 mg/ml/min) was topically applied for 3 min before topically administered IL-1beta (1microg/ml) and BK (10(-4) M). Monoclonal anti-mouse IL-1beta receptor antagonist (200 ng/ml), BK-B2 receptor antagonist (10(-6) M), PAF inhibitor (10(-5) M) and cycloheximide (10 microg/ml) were added topically 15, 10, 15 and 60 min, respectively, before IL-1beta (1 microg/ml). RESULTS: IL-1beta caused a significant increase in microvascular permeability, a decrease in capillary RBC velocity followed by increased leukocyte adhesion in postcapillary venules. BK caused a marked increase in leukocyte adhesion and no decrease in PCL and RBC velocity. Treatment with KLS significantly inhibited both the leukocyte adhesion and microvascular leakage induced by the two mediators. The inflammatory effects induced by IL-1beta were reduced by blockade of IL-1beta receptors and by a BK-B2 receptor antagonist but were not affected by a PAF antagonist and protein synthesis inhibition. CONCLUSIONS: These results demonstrate that KLS is effective in preventing early inflammatory changes induced by both IL-1beta and BK in the capillary network. Prostaglandin release and BK are essential components for IL-beta mediated responses, whereas neither PAF nor new protein synthesis appear to be linked to the early inflammatory changes induced by IL-1beta.     

Liquid chromatography-mass spectrometry assay of a thiadiazole derivative in mice: application to pharmacokinetic studies

Modern atmospheric pressure ionization (API) ion-trap mass spectrometry in connection with fast chromatographic separations using a short narrow-bore C8 column was developed to determine 5-phenyl-3-thioureido-1,2,4-thiadiazole (301029), a novel virus inhibitor in serum. Both 301029 and an internal standard (I.S.) were separated from serum samples by acetonitrile deproteinization and extraction without time-consuming reconstitution. The chromatographic separation was achieved on a C8 reversed-phase narrow-bore column using acetonitrile-water-acetic acid (90:10:0.01, v/v/v) as a mobile phase. The mass spectrometric analysis was performed by atmospheric pressure chemical ionization (APCI) mode with positive ion detection. Single ion monitoring (SIM) scan mode of m/z 237 and 158 was used to quantitatively determine 301029 and I.S., respectively. The low limit of quantitation was 25 ng/ml. The assay exhibited a linear range of 25-2500 ng/ml. Recovery from serum proved to be 100-113%. The precision (C.V.) and accuracy (RE) of the method were 2-12% and 94-112%, respectively. The present method was applied to determine the pharmacokinetic parameters of 301029 following oral administration of the agent to mice at 5 g/kg. The results revealed that the elimination half-life of 301029 was 413 min and the area under serum concentration-time curve was 354 microg/ml/min.     

High-performance liquid chromatographic determination of morphine and its 3- and 6-glucuronide metabolites by two-step solid-phase extraction

To provide more accurate measurement of morphine and its metabolites for a study of the genetic differences on morphine response, a method for the analysis of morphine and its metabolites is described which has the advantages of increased sensitivity and specificity by using a cleaner extraction. The new extraction method involves both the hydrophobic isolation on a carbon cartridge and ion-exchange isolation on ion-exchange resin which has not preliminary been described for morphine analysis. The combination of these two steps successfully purified drugs from human plasma with maximum removal of interfering substance comparing with a conventional C18 cartridge alone. The analytes are quantified by high-performance liquid chromatography on a reversed-phase C18 column employing a mobile phase consisting of 25% acetonitrile in 0.05 M phosphate buffer (pH 2.1), and 2.5 mM sodium dodecyl sulfate as the pairing ion with a combination of electrochemical and fluorometric detections. The recoveries for morphine (M), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and hydromorphone after the SPE procedure were 86+/-7.1%, 82+/-6.9%, 79+/-6.0% and 85+/-6.0%, respectively. Limits of detection for this method are 0.1 ng/ml for M, and 0.18 ng/ml for M3G and M6G. Limits of quantitation were approximately 0.25 ng/ml for M, and 0.45 ng/ml for M3G and M6G. The present assay was applied to measure M, M3G and M6G content in human plasma to test the applicability and suitability of this method for clinical and research use.     

Acute and chronic effects of vitamin C on endothelial fibrinolytic function in overweight and obese adult humans

We determined the effects of acute intra-arterial vitamin C administration and chronic oral vitamin C supplementation on the capacity of the endothelium to release t-PA in overweight and obese adults. Net endothelial t-PA release was determined in vivo in response to intrabrachial infusions of bradykinin and sodium nitroprusside in 33 sedentary adults: 10 normal-weight (BMI: 23.4 +/- 0.5 kg m(-2); 7M/3F); and 23 overweight/obese (BMI: 31.2 +/- 0.8 kg m(-2); 15M/8F). In 10 normal weight and eight overweight/obese adults the dose-response curves to bradykinin and sodium nitroprusside were repeated with a coinfusion of the antioxidant vitamin C (24 mg min(-1)). Seventeen of the 23 overweight/obese adults completed a 3 month chronic oral vitamin C (500 mg day(-1)) supplementation intervention. Intra-arterial administration of vitamin C significantly potentiated t-PA release in overweight/obese adults. Net release of t-PA was approximately 95% higher (P < 0.01) after (from -0.9 +/- 1.1 to 94.6 +/- 16.2 ng (100 ml tissue)(-1) min(-1)) compared with before (from -0.8 +/- 0.8 to 49.9 +/- 7.7 ng (100 ml tissue)(-1) min(-1)) vitamin C administration. Daily vitamin C supplementation significantly increased t-PA release in overweight/obese adults (from 0.2 +/- 0.9 to 48.2 +/- 6.5 ng (100 ml tissue)(-1) min(-1)) before supplementation versus (0.3 +/- 0.5 to 66.3 +/- 8.7 ng (100 ml tissue)(-1) min(-1)) after supplementation. These results indicate that the antioxidant vitamin C favourably affects the capacity of the endothelium to release t-PA in overweight/obese adults. Daily vitamin C supplementation represents an effective lifestyle intervention strategy for improving endothelial fibrinolytic regulation in this at-risk population.     

Development of a simplified, sensitive high-performance liquid chromatographic method using fluorescence detection to determine the concentration of UCN-01 in human plasma.

UCN-01 is a naturally derived anticancer agent isolated in the culture broth of actinomyces streptomyces. We have developed a sensitive high-performance liquid chromatographic method for the determination of UCN-01 in human plasma. UCN-01 was isolated from human plasma after intravenous administration, by using 100% ice-cold acetonitrile liquid-liquid phase extraction. Liquid chromatographic separation was achieved by isocratic elution on a phenyl analytical column. The mobile phase consisted of acetonitrile-0.5 M ammonium acetate (45:55) with 0.2% triethylamine added as a modifier. The UCN-01 peak was identified from other peaks using fluorescence excitation energy and emission energy wavelengths of 310 and 410 nm, respectively. Retention time for UCN-01 was 4.2 +/- 0.5 min. The UCN-01 peak was baseline resolved, with nearest peak at 2.6 min distance. No interfering peaks were observed at the retention time of UCN-01. Peak area amounts from extracted samples were proportional over the dynamic concentration range used: 0.2 to 30 microg/ml. Mean recoveries of UCN-01 at concentrations of 0.5 and 25 microg/ml were 89 and 90.2%, respectively. Relative standard deviations for UCN-01 calibration standards ranged from 1.89 to 2.31%, with relative errors ranging from 0.3 to 11.6%. Assay precision for UCN-01 based on quality control samples of 0.50 microg/ml was +/- 4.86% with an accuracy of +/-5.7%. For drug extracted from plasma the lowest limit of detection was 0.1 microg/ml, with the lowest limit of quantitation being 0.2 microg/ml. This method is suitable for routine analysis of UCN-01 in human plasma at concentration from 0.2 to 30 microg/ml.     

High sensitivity simultaneous determination in hair of the major constituents of ecstasy (3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylene-dioxyethylamphetamine) by high-performance liquid chromatography with direct fluorescence detection

A simple, but sensitive and specific high-performance liquid chromatographic assay for the simultaneous determination of the major constituents of "ecstasy" [i.e. 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxyethylamphetamine (MDE)] with direct fluorimetric detection, particularly intended for the routine analysis of hair, is described. Hair samples (100 mg) were overnight incubated in 1 ml of 0.25 M HCl at 45 degrees C and extracted with a commercial liquid-liquid method. The dried residue reconstituted with 500 microl of 0.05 M NaH2PO4 pH 5.2 was injected. Isocratic reversed-phase liquid chromatography was carried out on a column (250x4.6 mm I.D.) packed with spherical 5-microm poly(styrene-divinylbenzene) particles; the mobile phase was composed of 0.1 M potassium phosphate (pH 3)-acetonitrile (82:18). The excitation and the emission wavelengths were set to 285 and 320 nm, respectively. Under the described conditions, MDA, MDMA and MDE eluted in symmetric peaks with an analysis time of 30 min. The limit of detection was lower than 1 ng/ml, with a signal-to-noise ratio of 5, for each compound in solution, allowing a cut-off of 0.1 ng/mg in the hair matrix to be established. The intra-day precision (n = 6) of the assay was characterised by RSDs between 1.0 and 3.0% and between 0.52 and 0.88% for concentrations of 10 and 100 ng/ml, respectively; in day-to-day precision tests (n = 6), RSDs ranged between 5.12 and 11.12%, respectively, for the same concentrations. Interferences from as many as 92 therapeutic and/or abused drugs currently in use in the population were excluded, including N-methyl-1-(3,4-methylenedioxyphenyl)-2 butanamine (MBDB).     

Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites.

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C18 analytical column (10 cm x 0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran-acetonitrile-water (10:10:80, v/v) changing to tetrahydrofuran-acetonitrile-water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 microg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [14C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.     

Determination of a chemopreventive agent,Oltipraz, in rat plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a chemopreventive agent, Oltipraz, in rat plasma and urine. The sample preparation was simple; 2 volumes of acetonitrile were added to deproteinize the biological sample. A 50-microl aliquot of the supernatant was injected onto a C18 reversed-phase column. The mobile phase, acetonitrile : 0.5 mM ammonium acetate (55: 45, v/v for rat plasma and 45 : 55, v/v for rat urine), was run at a flow-rate of 1.5 ml/min. The column effluent was monitored using an ultraviolet detector set at 305 nm. The retention times for Oltipraz in rat plasma and urine were approximately 5.8 and 8.6 min, respectively. The detection limits of Oltipraz in rat plasma and urine were 20 and 50 ng/ml, respectively. The coefficients of variation of the assay (within-day and between-day) were generally low (below 4.65%) in concentration ranges from 0.02 (0.05) to 10 microg/ml for rat plasma and urine. No interference from endogenous substances was found.     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cmX4.6 mm I.D.) column, with mobile phase of acetonitrile-ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min(-1). Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05-10.0 microg ml(-1) in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 microg ml(-1) to 2.9% and 3.3% at 10.0 microg ml(-1) for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 microg ml(-1) to 7.6% and 11.5% at 10.0 microg ml(-1) for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4 +/- 1.77% and 76.1 +/- 7.26% for AQ4. The limit of detection was 50 ng ml(-1) with a 100 microl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Improved coupled column liquid chromatographic method for high-speed direct analysis of urinary trans,trans-muconic acid, as a biomarker of exposure to benzene

A coupled column liquid chromatographic (LC-LC) method for high-speed analysis of the urinary ring-opened benzene metabolite, trans,trans-muconic acid (t,t-MA) is described. Efficient on-line clean-up and concentration of t,t-MA from urine samples was obtained using a 3 microm C18 column (50x4.6 mm I.D.) as the first column (C-1) and a 5 microm C18 semi-permeable surface (SPS) column (150x4.6 mm I.D.) as the second column (C-2). The mobile phases applied consisted, respectively, of methanol-0.05% trifluoroacetic acid (TFA) in water (7:93, v/v) on C-1, and of methanol-0.05% TFA in water (8:92, v/v) on C-2. A rinsing mobile phase of methanol-0.05% TFA in water (25:75, v/v) was used for cleaning C-1 in between analysis. Under these conditions t,t-MA eluted 11 min after injection. Using relatively non-specific UV detection at 264 nm, the selectivity of the assay was enhanced remarkably by the use of LC-LC allowing detection of t,t-MA at urinary levels as low as 50 ng/ml (S/N>9). The study indicated that t,t-MA analysis can be performed by this procedure in less than 20 min requiring only pH adjustment and filtration of the sample as pretreatment. Calibration plots of standard additions of t,t-MA to blank urine over a wide concentration range (50-4000 ng/ml) showed excellent linearity (r>0.999). The method was validated using urine samples collected from rats exposed to low concentrations of benzene vapors (0.1 ppm for 6 h) and by repeating most of the analyses of real samples in the course of measurement sequences. Both the repeatability (n=6, levels 64 and 266 ng/ml) and intra-laboratory reproducibility (n=6, levels 679 and 1486 ng/ml) were below 5%.     

In vitro activities of broad-spectrum cephalosporins against nonmeningeal isolates of Streptococcus pneumoniae: MIC interpretation using NCCLS M100-S12 recommendations

Publication of the NCCLS M100-S12 document in January 2002 introduced ceftriaxone and cefotaxime MIC interpretative breakpoints of < or =1 microg/ml (susceptible), 2 microg/ml (intermediate), and > or =4 microg/ml (resistant) for nonmeningeal isolates of Streptococcus pneumoniae. To estimate the effect of these breakpoint changes on clinical laboratory susceptibility testing results, nonmeningeal pneumococcal isolate (blood and respiratory) data from The Surveillance Network Database-USA, an electronic surveillance database, for the years 1996 to 2000 were collated and studied. Of 9,863 nonmeningeal isolates tested against ceftriaxone, 82.7% were susceptible, 13.2% were intermediate, and 4.1% were resistant by the M100-S11 NCCLS breakpoints (2001); by M100-S12 breakpoints, 95.9% of the isolates were susceptible, 3.1% were intermediate, and 1.0% were resistant. Of 10,777 nonmeningeal isolates tested against cefotaxime, 79.2% were susceptible, 14.3% were intermediate, and 6.5% were resistant by M100-S11 breakpoints; by M100-S12 breakpoints, 93.5% were susceptible, 4.2% were intermediate, and 2.3% were resistant. Overall, the new M100-S12 ceftriaxone and cefotaxime interpretative breakpoints for nonmeningeal isolates of S. pneumoniae decreased the number of isolates interpreted as intermediate by 10% and as resistant by 3 to 4%.     

Rapid, highly sensitive method for the determination of morphine and its metabolites in body fluids by liquid chromatography-mass spectrometry

A rapid, highly sensitive method for the determination of morphine and its metabolites morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and normorphine has been developed using high-performance liquid chromatography-electrospray mass spectrometry, with the deuterated analogues as internal standards. The analytes were extracted automatically using end-capped C2 solid-phase extraction cartridges. Baseline separation of morphine, M3G and M6G was achieved on a LiChrospher 100 RP-18 end-capped analytical column (125x3 mm I.D., 5 microm particle size) with water-acetonitrile-tetrahydrofuran-formic acid (100:1:1:0.1, v/v) as the mobile phase. Morphine and normorphine coeluate and were separated mass spectrometrically. The mass spectrometer was operated in the selected-ion monitoring mode using m/z 272 for normorphine, m/z 286 for morphine, m/z 462 for morphine-6-glucuronide. Due to an interfering peak, M3G was measured by tandem mass spectrometry in the daughter-ion mode. The limits of quantitation achieved with this method were 1.3 pmol/ml for morphine, 1.5 pmol/ml for normorphine, 1.0 pmol/ml for M6G and 5.4 pmol/ml for M3G in serum or cerebrospinal fluid. The limits of quantitation achieved in urine were 10 pmol/ml for morphine, 20 pmol/ml for normorphine and M6G and 50 pmol/ml for M3G using a sample size of 100 microl. The method described was successfully applied to the determination of morphine and its metabolites in human serum, cerebrospinal fluid and urine in pharmacokinetic and drug interaction studies.     

Development of liquid chromatographic method for the analysis of kanamycin residues in varicella vaccine using phenylisocyanate as a derivatization reagent

A liquid chromatographic method for the determination of the aminoglycoside kanamycin in varicella vaccine is described. Kanamycin sulfate was derived with phenylisocyanate (PIC) and triethylamine for 10 min at 70 degrees C and chromatographed on a alkylamide-bonded column, Suplex pKb-100. A derivative of kanamycin sulfate was attached to four phenylisocynato groups and that molecular mass was confirmed with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). The kanamycin-PIC derivative was found to have a retention time of 11.7 min using an eluent composed of 40% acetonitrile in water at 1.2 ml/min column flow-rate. Detection was at a wavelength of 240 nm. Recoveries ranging from 97.5 to 99.8% were found. The correlation coefficient was greater than 0.9998 over the range between 10 and 100 microg/ml. The method precision of within-day assay showed a 0.5 to 4.0% coefficient of variation (n = 5) ranging from 10 to 70 microg/ml of kanamycin concentration levels. Kanamycin-PIC derivative in reaction solution was stable for 24 h at room temperature. A simple and efficient method for the analysis of the kanamycin in varicella vaccine was developed and validated.