Opioidergic control of gonadotropin secretion in the female rabbit: divergent effects of morphine on secretion of follicle-stimulating hormone and luteinizing hormone

The nature of secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was followed in female rabbits on a daily basis from age 36 to 60 days by sequential 5-min blood sampling over 1- to 2-h periods each day. Both LH and FSH were found to be secreted in a pulsatile manner. The mean LH pulse amplitude over the 25 days was 0.95 +/- 0.32 ng/mL and for FSH it was 10.15 +/- 1.11 ng/mL. Mean plasma LH levels were significantly increased from 1.46 +/- 0.08 ng/mL in 36 to 42-day-old rabbits to 1.89 +/- 0.12 ng/mL in 43 to 50-day-old rabbits and remained elevated from 50 to 60 days. FSH levels during the same periods also rose significantly from 14.93 +/- 0.79 to 19.57 +/- 2.05 ng/mL. To examine the influence of endogenous opioid peptides on the release of LH and FSH in 36 to 60-day-old female rabbits, morphine sulfate at 0.2, 0.5, 2.0, and 5.0 mg/kg was administered subcutaneously after 30 min baseline sampling, and blood was taken for another 60-120 min. Morphine at all doses and at all ages inhibited the amplitude and frequency of LH pulses but had no effect on FSH secretion. To determine whether the effects of morphine on LH secretion could be reversed with naloxone, females aged 82-114 days were used. Naloxone administered 1 h after morphine reversed the inhibitory effects of morphine, whereas the simultaneous administration of naloxone with morphine had variable effects but seemed to delay the LH increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and validation of a simple, sensitive enzyme immunoassay (EIA) for quantification of prolactin in buffalo plasma

A simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for prolactin quantification in buffalo plasma (on a microtitreplate) using the biotin-streptavidin-peroxidase amplification and immobilized antiserum in a competitive assay. Prolactin standards (range: 5-5000 pg/(well 50 microL)) were prepared in hormone-free plasma collected from minimal stress non-lactating buffalo heifers in temperate weather. The sensitivity of the EIA procedure was 5 pg/(well 50 microL) (corresponds to 0.1 ng/mL plasma); the 50% relative binding sensitivity occurred at 160 ng/(well 50 microL). Plasma volumes for the EIA, viz. 12.5, 25, and 50 microL, did not influence the shape of standard curve. A parallelism test was carried out to compare the endogenous buffalo plasma prolactin with bovine prolactin standard. To validate the assay biologically, 11 Murrah buffaloes were given a third-generation antiprolactin (Norprolac; 10 mg/animal, i.m.). Blood samples were collected 1 d prior to the start of Norprolac administration and continued up to seventh day in an Ovsynch treatment program. In all animals, there were abrupt declines in prolactin concentrations following Norprolac treatments, which confirmed the biological validation of the EIA. After development and validation of EIA procedure, the concentration of plasma prolactin was determined efficiently in samples collected during both summer and winter samples.     

Transfer of morphine across the human placenta and its interaction with naloxone

The purpose of this investigation was to measure the transfer rate and clearance of morphine across the placenta with and without naloxone. Term human placental cotyledons were perfused in vitro. The placenta was perfused with 50 ng/mL of morphine in the absence (n=4) and presence (n=5) of 100 ng/mL of naloxone. Maternal and fetal samples were collected. Student's t-test or one-way repeated measures ANOVA were used for all comparisons. The maternal-to-fetal morphine transfer rate was 0.73+/-0.44 ng/mL/min in the morphine and 0.69+/-0.26 ng/mL/min in the morphine-naloxone experiments (p=0.89). The clearance of morphine was 0.89+/-0.39 mL/min without naloxone and 0.87+/-0.27 mL/min with naloxone (p=0.92). Final morphine concentrations in the morphine experiments were 9.78+/-6.17 ng/mL (maternal) and 3.43+/-2.14 ng/mL (fetal) and 10.04+/-3.89 ng/mL (maternal) and 4.16+/-1.64 ng/mL (fetal) in the morphine-naloxone experiments. Morphine readily crosses the term human placenta. Naloxone does not alter placental transfer or clearance of morphine, suggesting that transfer across the placental barrier is not altered by changes in vascular resistance. Placental retention of morphine prolongs fetal exposure to morphine.     

A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry

A sensitive, specific and fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay for the determination of vinorelbine in mouse and human plasma is presented. A 200 microL aliquot was extracted with solid-phase extraction (SPE) using Bond-Elut C(2) cartridges. Dried extracts were reconstituted in 100 microL 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) containing the internal standard vintriptol (100 ng/mL) and 10 microL volumes were injected onto the HPLC system. Separation was achieved on a 50 mm x 2.0 mm i.d. Gemini C(18) column using isocratic elution with 1 mM ammonium acetate pH 10.5-acetonitrile-methanol (21:9:70, v/v/v) at a flow rate of 0.4 mL/min. HPLC run time was only 5 min. Detection was performed using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay quantifies vinorelbine from 0.1 to 100 ng/mL using human plasma sample volumes of 200 microL. With this method vinorelbine can be measured in mouse plasma samples when these samples are diluted eight times in control human plasma. Calibration samples prepared in control human plasma can be used for the quantification of the drug. The lower limit of quantification in mouse plasma is 0.8 ng/mL. This assay is used to support preclinical and clinical pharmacologic studies with vinorelbine.     

Cardiopulmonary effects of nalbuphine hydrochloride and butorphanol tartrate in sheep

The cardiopulmonary, sedative and analgesic effects of butorphanol tartrate and nalbuphine hydrochloride were evaluated in six adult crossbred Dorset sheep (Ovis aries). The animals were divided randomly into two groups of three. The first group received butorphanol tartrate (0.5mg/Kg s.c.) followed in 3 days by nalbuphine hydrochloride (1 mg/Kg, s.c.). The second group received nalbuphine followed in 3 days by butorphanol. Cardiopulmonary parameters were evaluated at baseline (once the animal had accommodated to restraint); immediately following analgesic administration; and at 15, 30, 60, 90 and 120 minutes after analgesic administration. No significant changes (alpha greater than .05) from baseline were seen in any of the measured cardiopulmonary parameters from either the butorphanol or nalbuphine groups. Butorphanol produced the most dramatic analgesic and sedative effects with onset of both within 15 minutes of administration and peak effects occurring 30 minutes post injection. The degree of analgesia was diminished at 120 minutes while the sedative effect returned to near baseline by 90 minutes. The nalbuphine group also showed an onset of analgesia 15 minutes post injection reaching a peak effect after 30 minutes. However, onset of sedation occurred 30 minutes post injection achieving a peak effect at 60 minutes which was markedly less than that of butorphanol. As in the butorphanol group, analgesia was diminished at 120 minutes.     

The effects of morphine and various narcotic antagonist type analgesics on body temperature in rats

ED50s were determined for morphine, nalorphine, butorphanol and pentazocine induced hyperthermia in rats. Morphine produced a significant hyperthermia with the doses of 5–160 mg.kg in rats. The peak hyperthermic effect was found 1 hr after 5–20 mg/kg doses of morphine. Nalorphine, butorphanol and pentazocine produced biphasic effects on rectal temperature. Initially they produced a dose-dependent hyperthermia and later hypothermia. In a comparison of the hyperthermic ED50's of morphine, nalorphine, butorphanol and pentazocine it was found that butorphanol is more active than the others (ED50s were 4.7, 4.3, 0.54 and 11.5 mg/kg respectively). The narcotic antagonist naloxone significantly inhibited both morphine and antagonist type analgesic induced hyperthermia. These results suggests that a different mechanism(s) is involved in the hyperthermic actions of antagonist type analgesics and agonist drugs.     

Liquid chromatographic method with amperometric detection to determine acteoside in rat blood and brain microdialysates and its application to pharmacokinetic study

A simple and sensitive liquid chromatography with amperometric detection was developed for the first time to monitor the protein-unbound acteoside in the rat blood and brain microdialysate by microdialysis technique. Microdialysis samples without further cleanup procedures were directly injected into the HPLC and separated using a reversed-phase C18 column (150 mmx2 mm, i.d. 5 microm) maintained at ambient temperature and a mobile phase comprised of acetonitrile-50 mM monosodium phosphate (pH 2.8) (17:83, v/v) with a flow rate of 0.2 mL/min. Based on the experimental voltamogram, the applied potential was set at +0.9 V oxidative mode. The concentration-response relationship was linear (r2>0.99) over a concentration range of 5-500 ng/mL; method precision and accuracy fell within predefined limits (less than 20%). The developed method was applied to assess the pharmacokinetics of acteoside, and the results suggested that acteoside was fitted better by the two-compartmental model following a single intravenous injection of acteoside. Acteoside was unable to be detected in the brain dialysate. The distribution and elimination half-lives of unbound acteoside in the blood were 5 and 28 min, respectively, which suggested the rapid distribution of acteoside.     

Determination of butorphanol in horse race urine by immunoassay and gas chromatography–mass spectrometry

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC–MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC–MS system. The ELISA test (20 μl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC–MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC–MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10–250 ng/ml), recovery (±100%), intra-assay (4.1–14.9%) and inter-assay (9.3–45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).     

High-performance liquid chromatographic method for the simultaneous determination of nalbuphine and its prodrug, sebacoyl dinalbuphine ester, in dog plasma and application to pharmacokinetic studies in dogs

For the determination of nalbuphine and its long acting prodrug, sebacoyl dinalbuphine ester (SDN), in biological samples, a reversed-phase high-performance liquid chromatographic method using dual detectors was established. Ultraviolet and fluorescence detectors were connected in series for determining SDN and nalbuphine, respectively. The two analytes and internal standard were extracted from plasma by alkaline liquid–liquid extraction using n-hexane–isoamyl alcohol (9:1, v/v). The calibration curve for nalbuphine was linear over the range from 10 to 2500 ng/ml, while the range was 25 to 2500 ng/ml for SDN. The within- and between-day precision and accuracy were all within 10% for both nalbuphine and SDN over these concentrations. The method was applied successfully to a pharmacokinetic study of SDN administered at 20 mg/kg to two beagle dogs. Pharmacokinetic analysis revealed that SDN followed a linear one-compartment model with an elimination half-life of 74.7 min. Formation of nalbuphine after intravenous administration of SDN was observed in the first time point sample (5 min). These results indicate that SDN is rapidly metabolized to its active moiety, nalbuphine, in dogs and no other metabolites are detected in plasma.     

Rapid and simultaneous determination of tacrolimus (FK506) and diltiazem in human whole blood by liquid chromatography-tandem mass spectrometry: application to a clinical drug-drug interaction study

Tacrolimus (FK506) is a potent immunosuppressant widely used for organ transplantation patients while diltiazem (DTZ), a calcium-channel inhibitor, is often used in renal transplantation patients to prevent post-transplant hypertension. However, DTZ has a significant pharmacokinetic interaction with FK506. In this study, a rapid and sensitive ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of FK506 and DTZ in human whole blood using ascomycin as the internal standard (IS). After extraction of the whole blood samples by ethyl acetate, FK506, DTZ and the IS were subjected to LC/MS/MS analysis using electro-spray positive-ion mode ionization (ESI(+)). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The MS/MS detection was conducted by monitoring the fragmentation of 821.7-->768.9 (m/z) for FK506, 415.5-->310.3 (m/z) for DTZ and 809.8-->757.0 (m/z) for IS. The method had a chromatographic running time of approximately 2 min and linear calibration curves over the concentrations of 0.5-200 ng/mL for FK506 and 2-250 ng/mL for DTZ. The recoveries of liquid-liquid extraction method were 58.3-62.6% for FK506 and 50.4-58.8% for DTZ. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for FK506 and 2 ng/mL for DTZ. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL for FK506 and 5, 25, and 100 ng/mL for DTZ. The validated LC/MS/MS method has been successfully used to analyze the concentrations of FK506 and DTZ in whole blood samples from pharmacokinetic studies in renal transplanted patients.     

Quantification of cationic anti-malaria agent methylene blue in different human biological matrices using cation exchange chromatography coupled to tandem mass spectrometry

Selective and sensitive methods for the determination of the cationic dye and anti-malarial methylene blue in human liquid whole blood, dried whole blood (paper spot), and plasma depending on protein precipitation and cation exchange chromatography coupled to electrospray ionisation (ESI) tandem mass spectrometry (MS/MS) have been developed, validated according to FDA standards, and applied to samples of healthy individuals and malaria patients within clinical studies. Acidic protein precipitation with acetonitrile and trifluoroacetic acid was used for liquid whole blood and plasma. For the extraction of methylene blue from paper spots aqueous acetonitrile was used. Sample extracts were chromatographed on a mixed mode column (cation exchange/reversed phase, Uptisphere MM1) using an aqueous ammonium acetate/acetonitrile gradient. Methylene blue was quantified with MS/MS in the selected reaction monitoring mode using ESI and methylene violet 3RAX as internal standard. Depending on the sample volume (whole blood and plasma 250 microL, and 100 microL on paper spots) the method was linear at least within 75 and 10,000 ng/mL and the limit of quantification in all matrices was 75 ng/mL. Batch-to-batch accuracies of the whole blood, plasma, and paper spot methods varied between -4.5 and +6.6%, -3.7 and +7.5%, and -5.8 and +11.1%, respectively, with corresponding precision ranging from 3.8 to 11.8% CV. After a single oral dose (500 mg) methylene blue concentrations were detectable for 72 h in plasma. The methods were applied within clinical studies to samples from healthy individuals and malaria patients from Burkina Faso.     

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography-mass spectrometry

A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography-mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax/divinylbenzene (CW/DVB 65 microm) and polydimethylsiloxane (PDMS 100 microm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 microL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 microg/mL for blood, and 0.01 to 50 microg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within +/-15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.     

Reduction of the sevoflurane minimum alveolar concentration induced by methadone, tramadol, butorphanol and morphine in rats

This study aimed to estimate the reduction in the minimum alveolar concentration (MAC) of sevoflurane induced by low and high doses of methadone (5 and 10 mg/kg), tramadol (25 and 50 mg/kg), butorphanol (5 and 10 mg/kg) or morphine (5 and 10 mg/kg) in the rat. A control group received normal saline. Sixty-three adult male Sprague-Dawley rats were anaesthetized with sevoflurane (n = 7 per group). Sevoflurane MAC was then determined before and after intraperitoneal administration of the opioids or saline. The duration of the sevoflurane MAC reduction and basic cardiovascular and respiratory measurements were also recorded. The baseline MAC was 2.5 (0.3) vol%. Methadone, tramadol and morphine reduced the sevoflurane MAC (low dose: 31 ± 10, 38 ± 15 and 30 ± 13% respectively; high dose: 100 ± 0, 83 ± 17 and 77 ± 25%, respectively) in a dose-dependent manner. The low and high doses of butorphanol reduced the sevoflurane MAC to a similar extent (33 ± 7 and 31 ± 4%, low and high doses, respectively). Two rats developed apnoea following administration of high-dose butorphanol and methadone. These anaesthetic-sparing effects are clinically relevant and may reduce the adverse effects associated with higher doses of inhalational anaesthetics.     

Development and validation of a rapid and sensitive high-performance liquid chromatography-mass spectroscopy assay for determination of 17-(allylamino)-17-demethoxygeldanamycin and 17-(amino)-17-demethoxygeldanamycin in human plasma

A sensitive method was developed and validated for the measurement of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) and its active metabolite 17-amino-17-demethoxygeldanamycin (17AG) in human plasma using 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG) as an internal standard. After the addition of internal standard, 200 microL of plasma was extracted using ice cold acetonitrile followed by analysis on a Thermo Finnigan triple-quadruple mass spectrometer coupled to an Agilent 1100 HPLC system. Chromatography was carried out on a 50 mm x 2.1 mm Agilent Zorbax SB-phenyl 5 microm column coupled to a 3mm Varian metaguard diphenyl pre-column using glacial acetic acid 0.1% and a gradient of acetonitrile and water at a flow rate of 500 microL/min. Atmospheric pressure chemical ionization and detection of 17AAG, 17AG and 17DMAG were accomplished using selected reaction monitoring of m/z 584.3>541.3, 544.2>501.2, and 615.3>572.3, respectively in negative ion mode. Retention times for 17AAG, 17AG, and 17DMAG were 4.1, 3.5, and 2.9 min, respectively, with a total run time of 7 min. The assay was linear over the range 0.5-3000 ng/mL for 17AAG and 17AG. Replicate sample analysis indicated within- and between-run accuracy and precision within 15%. The recovery of 17AAG and 17AG from 200 microL of plasma containing 1, 25, 300, and 2500 ng/mL was 93% or greater. This high-performance liquid chromatographic tandem mass spectroscopy (HPLC/MS/MS) method is superior to previous methods. It is the first analytical method reported to date for the quantitation of both 17AAG and its metabolite 17AG and can reliably quantitate concentrations of both compounds as low as 0.5 ng/mL.     

Determination of lapatinib (GW572016) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive method for the determination of lapatinib (GW572016) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Plasma samples (100 microL) were prepared using solid phase extraction (SPE) columns, and 6.0 microL of the reconstituted eluate was injected onto a Phenomenex CuroSil-PFP 3 mu analytical column (50 mm x 2.0mm) with an isocratic mobile phase. Analytes were detected with a PE SCIEX API-365 LC-MS/MS system at unit (Q1) and low (Q3) resolution in positive multiple reaction monitoring mode (m/z 581 (precursor ion) to m/z 364 (product ion) for lapatinib). The mean recovery for lapatinib was 75% with a lower limit of quantification of 15 ng/mL (S/N=11.3, CV< or =14%). This method was validated over a linear range of 100-10,000 ng/mL, and results from a 5-day validation study demonstrated good within-day and between-day precision and accuracy. This method has been used to measure plasma lapatinib concentrations in a Phase I study in children with cancer.     

Development and validation of a selective and robust LC-MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: application to a clinical pharmacokinetic study

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of a potent 5-HT(1B/1D) receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 microL plasma samples by liquid-liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6mm x 50mm, 5 microm) with a mobile phase consisting of acetonitrile-10mM aqueous ammonium acetate-acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270-->201 (rizatriptan) and 313.4-->138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05-50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.     

High-performance liquid chromatographic method for the quantitative determination of butorphanol, hydroxybutorphanol, and norbutorphanol in human urine using fluorescence detection

A sensitive, quantitative reversed-phase high-performance liquid chromatographic method has been established for the simultaneous determination of butorphanol, a synthetic opioid, and its metabolites, hydroxybutorphanol and norbutorphanol, in human urine samples. The method involved extraction of butorphanol, hydroxybutorphanol, and norbutorphanol from urine (1.0 ml), buffered with 0.1 ml of 1.0 M ammonium acetate (pH 6.0), onto 1-ml Cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the HPLC mobile phase, acetonitrile—methanol—water (20:10:70, v/v/v), containing 10 mM ammonium acetate and 10 mM TMAH (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5-μm column. The analysis was accomplished by detection of the fluorescence of the three analytes, at excitation and emission wavelengths of 200 nm and 325 nm, respectively. The retention times for hydroxybutorphanol, norbutorphanol, the internal standard, and butorphanol were 5.5, 9.0, 13.0, and 23.4 min respectively. The validated quantitation range of the method was 1–100 ng/ml for butorphanol and hydroxybutorphanol, and 2–200 ng/ml for norbutorphanol in urine. The observed recoveries for butorphanol, hydroxybutorphanol, and norbutorphanol were 93%, 72%, and 50%, respectively. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of study samples. The method was applied on study samples from a clinical study of butorphanol, providing a pharmacokinetic profiling of butorphanol.     

Determination of MDMA and MDA in rat urine by semi-micro column HPLC-fluorescence detection with DBD-F and their monitoring after MDMA administration to rat.

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and alpha-phenylethylamine as an internal standard (IS). A sample (50 microL) of rat urine was added to 5 microL IS and 100 microL 100 mmol/L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 microL 75 mmol/L borate buffer (pH 8.5) and 50 microL 20 mmol/L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80 degrees C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN/CH3OH/10 mmol/L imidazole-HNO3 buffer (pH 7.0) (= 45:5:50, v/v/v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5-15 ng/mL detection limits at a S/N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg/kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13-160.1 and 0.17-10.9 microg/mL, respectively.     

Sensitive method for the quantitative determination of proguanil and its metabolites in rat blood and plasma by liquid chromatography-mass spectrometry

A sensitive, simple and fast liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for the determination of proguanil (PG) and its metabolites, cycloguanil (CG) and 1-(4-chlorophenyl)biguanide (4CPB), was developed and validated over a concentration range of 1-2000 ng/mL using only 50 microL of blood or plasma. After a simple solvent precipitation procedure, the supernatant was analysed directly by HPLC-MS/MS. Separation was achieved using an ethyl-linked phenyl reverse phase column with polar endcapping with an acetonitrile-water-formic acid gradient. Mass spectrometry was performed using a triple quadrupole mass spectrometer operating in positive electrospray ionization mode. The elution of PG (254.07-->169.99), CG (252.12-->195.02) and 4CPB (212.06-->153.06) was monitored using selected reaction monitoring. The three compounds and the internal standard (chloroproguanil) were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in blood and plasma. The limit of quantification of PG and CG was 1 ng/mL and 5 ng/mL for 4CPB in rat blood and plasma. The extraction efficiency of PG, CG and 4CPB from rat blood and plasma was higher than 73%. The intra- and inter-assay variability of PG, CG and 4CPB were within 12% and the accuracy within +/-5%. This new assay offers higher sensitivity and a much shorter run time over earlier methods.     

Determination of Cloretazine (VNP40101M) and its active metabolite (VNP4090CE) in human plasma by liquid chromatography electrospray tandem mass spectrometry (LC-ESI-MS/MS)

A sensitive method for the determination of Cloretazine (VNP40101M) and its metabolite (VNP4090CE) with an internal standard (ISTD) in human plasma was developed using high-performance liquid chromatographic separation with tandem mass spectrometric detection. Acidified plasma samples (500 microL) were prepared using solid phase extraction (SPE) columns, and 25 microL of the reconstituted sample was injected onto an Ascentis C18 HPLC column (3 microm, 5 cmx2.1 mm) with an isocratic mobile phase. Analytes were detected with an API-3000 LC-MS/MS System at unit (Q1) and low (Q3) resolution in negative multiple reaction monitoring mode: m/z 249.0 (precursor ion) to m/z 114.9 (product ion) for both Cloretazine (at 3.64 min) and VNP4090CE (at 2.91 min), and m/z 253.0 (precursor ion) to m/z 116.9 (product ion) for the ISTD. The mean recovery for Cloretazine (VNP40101M) and its metabolite (VNP4090CE) was greater than 87% with a lower limit of quantification of 1.0 ng/mL for Cloretazine (S/N=9.7, CV相似文献