Quantitation of the glycation intermediate 3-deoxyglucosone by oxidation with rabbit liver oxoaldehyde dehydrogenase to 2-keto-3-deoxygluconic acid followed by high-performance liquid chromatography

A simple and sensitive method for the detection of 3-deoxyglucosone was developed using oxidation with crude oxoaldehyde dehydrogenase to 2-keto-3-deoxygluconic acid followed by high-performance liquid chromatography (HPLC). Oxoaldehyde dehydrogenase was prepared from rabbit liver and partially characterized. 2-Keto-3-deoxygluconic acid produced from 3-deoxyglucosone by oxoaldehyde dehydrogenase was derivatized with 1,2-diamino-4,5-methylenedioxybenzene, and the fluorescent products were detected and quantitated by HPLC using a solvent containing borate. In the presence of borate, 2-keto-3-deoxygluconic acid was completely separated from N-acetylneuraminic acid. The detection limit of 3-deoxyglucosone was 2.5 pmol/injection (10 μl) at a signal-to-noise ratio of 3. This method was used to confirm the inhibitory effect of aminoguanidine on glycation.     

Simultaneous determination of methamphetamine and its metabolite, amphetamine, in urine using a high performance liquid chromatography column-switching method

We describe here a simple, precise, and highly sensitive method for the simultaneous determination of methamphetamine (MA) and amphetamine (AM) in urine using a high performance liquid chromatography (HPLC) column-switching method. A PK-2A (Shodex) column was used for extraction and deproteinization, and a CAPCELL PAK SCX semi-micro, polymer-coated cation-exchange column was employed for separation. The urine sample was mixed with an equal volume of borate buffer (0.1M, pH 9.4), and then 100 microl of the mixture was injected into the HPLC column. The column was switched for 6 min, and then 10 min later detection was performed at 210 nm. Recovery yields of the MA and AM spiked in the urine were 93.0-100.4% with a coefficient of variation of less than 1%. The calibration curves of MA and AM were in the range of 0.1-10 microg/ml with good linearity (r(2)=0.999), with the limit of qualification being 0.005 microg/ml. This method of using HPLC with column-switching can be used for both qualification and quantification of MA and its metabolite, AM, in urine, especially in forensic cases.     

Capillary electrophoresis-based nanoscale assays for monitoring ecto-5'-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT.     

Determination of tianeptine in human plasma using high-performance liquid chromatography with fluorescence detection

A new, selective and sensitive high-performance liquid chromatography (HPLC) method with fluorimetric detection was developed for the determination of tianeptine (TIA) in human plasma using solid phase extraction (SPE) procedures. The method is based on the derivatization of TIA with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer of pH 8.5 to yield a yellow, fluorescent product. The HPLC separation was achieved on a Phenomenex C(18) column (250 mm x 4.6 mm) using a mobile phase of acetonitrile-10mM orthophosphoric acid (pH 2.5) (77:23, v/v) solvent system at 1 mL/min flow rate. Gabapentin (GA) was used as the internal standard. The fluorometric detector was operated at 458 nm (excitation) and 520 nm (emission). The assay was linear over the concentration range of 5-300 ng/mL. The detection limit (LOD) was found to be 2 ng/mL. The mean recovery was determined to be 88.6%. The proposed method was applied for pharmacokinetic study of 12.5mg TIA in a healthy volunteer.     

Synthesis of chiral vicinal diols and analysis of them by capillary zone electrophoresis

This paper describes an improved access to 1,4-bis (9-O-quininyl) phthalazine [(QN)(2)PHAL], a very useful chiral ligand for catalytic asymmetric dihydroxylation (AD), by using CaH(2) as acid-binding reagent in a high yield under mild conditions. The application of (QN)(2)PHAL to the AD reactions of eight olefins exhibited excellent enantioselectivity and activity with corresponding chiral vicinal diols. Furthermore, a capillary zone electrophoresis method was developed to separate the aforementioned chiral vicinal diols by using of neutral beta-cyclodextrin (beta-CD) as chiral selector and borate as running buffer. High resolution was achieved under the optimal conditions of beta-CD 2.2% (w/v), pH 10, 200 mM borate buffer at 15 kV, and 20 degrees C within 15 min. The relative standard deviations of the corrected peak areas and migration time were less than 3.9% and 1.3%, respectively. In addition, the developed method was successfully applied to the determination of the purity and the enantiomeric excesses value (%ee) of the AD reaction products.     

毛脉酸模不同生长发育期HPLC色谱指纹图谱研究

采用HPLC-DAD方法,梯度洗脱,对10批不同生长发育期毛脉酸模根样品进行主成分分析。色谱条件为:Planetsil C18分析柱(5μm, 200 mm×4.6 mm),预柱Phenomenex ODS-C18(4×3.0 mm ID),柱温40℃,流动相A为甲醇;流动相B为水(磷酸调pH值为2.0),流动相A从30%甲醇到100%甲醇,时间为0~50 min,检测波长254 nm。10批毛脉酸模样品得到的色谱指纹图谱有27个共有峰,其特征峰指纹图谱可分为Ⅰ, Ⅱ, Ⅲ三组:第Ⅰ组包括0~17 min(1~14号峰),第Ⅱ组包括17~35 min(15~24号峰),第Ⅲ组包括35~50 min(25~27号峰)。HPLC-DAD方法分析毛脉酸模主成分,方法准确可靠,其本身具有多成分同时定性的优势;27个共有峰的出峰先后顺序及相对含量极具特征性、专属性,重复性好,形成了毛脉酸模特有的HPLC色谱指纹图谱,可作为毛脉酸模内在质量评价、鉴定及其最佳采收期确定提供科学依据。     

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.     

Determination of norepinephrine and its metabolites released from rat vas deferens using high-performance liquid chromatography with electrochemical detection

We designed a rapid, simple and sensitive method for the determination of norepinephrine (NE) and its metabolites by reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection. NE, 3,4-dihydroxymandelic acid (DOMA), and 3,4-dihydroxyphenylglycol (DOPEG) were adsorbed on alumina and eluted with 0.2 N HCl. From the remaining solution, normetanephrine and 3-methoxy-4-hydroxyphenylglycol (MOPEG) were extracted with ethyl acetate in the presence of both borate buffer and K₂HPO₄. Vanillylmandelic acid was extracted with ethyl acetate after acidification of the solution with concentrated HCl. The combined ethyl acetate phase was evaporated and the residue was dissolved in 0.1 N HCl. A 50 μl aliquot of each eluate or solution was injected onto the HPLC. Detection limits ranged from 300 pg to 1 ng per initial sampla. We used this method to determine substances in the medium following incubation of the rat vas deferens. Approximately 110 and 80 ng/g/10 min of DOPEG and MOPEG, respectively, were present under normal conditions. The electrical stimulation of tissues from the rat vas deferens led to increases in the levels of NE, DOPEG, DOMA and MOPEG. Normetanephrine and vanillylmandelic acid were not detected in the medium. This is probably the first documentation of the endogenous levels of NE and all its metabolites in medium containing tissue of the sympathetic nervous system.     

Determination of the dissociation constants of ropinirole and some impurities and their quantification using capillary zone electrophoresis

Ropinirole, 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one, is a potent anti-Parkinson’s disease drug developed by SmithKline Beecham Pharmaceuticals. Capillary zone electrophoresis (CZE) was used for the determination of the dissociation constants of ropinirole and five structurally related impurities, potentially formed during its synthesis and for separation and quantification of these substances. The dissociation constants obtained from the CZE measurements were confirmed by UV spectrophotometry for some of the test compounds, obtaining a good agreement between the values. Careful optimization of the running buffer composition permitted base-line resolution of the six compounds in a borate buffer containing acetonitrile and magnesium sulfate (a 100 mM borate buffer containing 30 mM MgSO₄ and 20 vol.% of acetonitrile). It was shown that CZE can determine the level of these impurities, down to a level of 0.05% of the main component within 15 min.     

Manganese-dependent inhibition of human liver arginase by borate

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.     

Separation of bisbenzylisoquinoline alkaloids by micellar electrokinetic chromatography

The micellar electrokinetic chromatographic (MEKC) separation of seven bisbenzylisoquinoline alkaloids has been developed. The effects of various separating factors were studied. Optimum separation was achieved using a buffer (pH 9.2) of 20 mM sodium borate and 20 mM sodium dihydrogen phosphate buffer containing 55 mM sodium cholate; the optimum voltage and injection time were 21 kV and 0.05 min, respectively. Highest peak efficiency was obtained when the analytes were dissolved in 10 mM sodium dodecyl sulphate as sample matrix for injection. The elution order of the bisbenzylisoquinoline alkaloids was related to their lipophilicity. The resolution, run time and detection limits of the MEKC method were compared with those of an HPLC method developed previously.     

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.     

Phenotype of CYP2C19 and CYP3A4 by determination of omeprazole and its two main metabolites in plasma using liquid chromatography with liquid-liquid extraction

We present a new simple and reliable HPLC method for measuring omeprazole and its two main metabolites in plasma. This can be used for studying CYP2C19 and CYP3A4 genetic polymorphisms using omeprazole as the probe drug. Omeprazole, hydroxyomeprazole and omeprazole sulfone were extracted from plasma samples with phosphate buffer and dichloromethane-ether (95:5). HPLC separation was achieved using an Ultrasphere ODS C(18) (Beckman) column. The mobile phase was acetonitrile-phosphate buffer (24:76, pH 8), containing nonylamine at 0.015%. Retention times were 9.5 min for omeprazole, 3.25 min for hydroxyomeprazole, 7.4 min for omeprazole sulfone and 6.27 min for internal standard (phenacetine). Detection (UV at 302 nm) of analytes was linear in the range from 96 to 864 ng/ml. This is useful for calculating metabolic index for CYP2C19 and CYP3A4 in adults and children. This method is stable, reproducible, improves resolution and has practical advantages such as low cost.     

A sensitive and reproducible assay to measure the activity of glucosylceramide synthase and lactosylceramide synthase using HPLC and fluorescent substrates

Glucosylceramide synthase (GlcT) and lactosylceramide synthase (GalT) are key enzymes for the synthesis of major glycosphingolipids of vertebrates. In this article, we report a new reliable method to determine GlcT and GalT activities using the fluorescent acceptor substrates C6-4-nitrobenzo-2-oxa-1,3-diazole (NBD)-ceramide and C6-NBD-glucosylceramide, respectively, and a normal-phase high-performance liquid chromatography (HPLC). The reaction products, C6-NBD-glucosylceramide for GlcT and C6-NBD-lactosylceramide for GalT, could be separated from the corresponding acceptor substrates within 6 min under the conditions used. Reaction products were able to be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol, making it possible to determine both activities using the lysate from 1 x 10(4) cultured CHOP cells (Chinese hamster ovary cells expressing polyoma LT antigen) and one zebrafish embryo. This method was used successfully to evaluate the degree of knockdown of GlcT and GalT during zebrafish embryogenesis after injection of the morpholino-oligo-based antisense into one- to four-cell embryos. These results indicate that the fluorescence-based HPLC method is a highly sensitive, rapid, and reproducible assay for determining GlcT and GalT activities and is useful for evaluating the activities in gene knockdown experiments.     

IL-1 increases phospholipase A2 activity, expression of phospholipase A2-activating protein, and release of linoleic acid from the murine T helper cell line EL-4.

The early events in IL-1-mediated activation of T cells were investigated in the murine T cell line, EL-4. Treatment of EL-4 cells with human rIL-1 beta resulted in a rapid increase in phospholipase A2 (PLA2) activity. PLA2 activity increased approximately fivefold within 4 min after exposure to IL-1. Synthesis of the phospholipase A2- activating protein (PLAP) and its mRNA were also increased within 4 min of IL-1 treatment and preceded the increase in PLA2 enzyme activity. The increases in PLA2 activity and PLAP protein and mRNA levels were all transient and declined to baseline within 10 min after the addition of IL-1. The changes in levels of PLAP as a function of time after IL-1 treatment were consistent with PLAP playing an important role in the regulation of PLA2 activity in this system. The consequence of the elevated PLA2 activity was examined by analysis of the fatty acids released from IL-1-treated cells. There was a 20-fold increase in the release of radioactivity from [14C]-linoleic acid labeled cells whereas there was very little change in the release of radioactivity from [14C]-arachidonic acid labeled cells in response to the addition of IL-1. The radioactivity released from [14C]-linoleic acid labeled cells was analyzed by HPLC; no conversion of radiolabeled linoleic into arachidonic acid was observed. In EL-4 cells, IL-1 potentiates PMA-mediated release of IL-2 at suboptimal concentrations of PMA. Linoleic acid also augmented PMA-induced IL-2 release from the EL-4 cells. This fatty acid was more than 10 times more effective than arachidonic acid in this regard. Furthermore, the addition of exogenous PLAP to EL-4 cells could substitute for IL-1 in the stimulation of IL-2 release. These results suggest that the IL-1 effects on T cells may be mediated at least in part through increased PLA2 activity due to increased synthesis of PLAP. Furthermore, the release of the unsaturated fatty acid linoleic acid or its metabolites may be of functional importance in IL-1-mediated IL-2 production by EL-4 cells.     

Quantification of 1,5-anhydro-d-glucitol in urine by automated borate complex anion-exchange chromatography with an immobilized enzyme reactor

HPLC using a borate form of a strongly anion-exchange resin column and an immobilized enzyme reactor for colorimetric detection was used to quantify urinary 1,5-anhydro-d-glucitol. Urine samples were introduced into the system every 7 min without any pretreatment, and after separation of interfering substances in the column, 1,5-anhydro-d-glucitol was successively detected. Quantitative determination of urinary 1,5-anhydro-d-glucitol was possible within the 1.2–300 μmol/l range. The coefficient of variance was less than 3% and the correlation between results obtained with our system (y) and those obtained by gas chromatography–mass spectrometry (x) was y=0.983x−1.287 μmol/l (n=42, r=0.998).     

Simultaneous determination of deoxyribonucleoside in the presence of ribonucleoside triphosphates in human carcinoma cells by high-performance liquid chromatography.

Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in cells by HPLC is an analytical challenge since the concentration of dNTP present in mammalian cells is several orders of magnitude lower than the corresponding NTP. Hence, the quantitation of dNTP in cells is generally performed after selective oxidation or removal of the major NTP. The procedures reported so far are lengthy and cumbersome and do not enable the simultaneous determination of NTP. We report the development of a simple, direct HPLC method for the simultaneous determination of dNTP and NTP in colon carcinoma WiDr cell extracts using a stepwise gradient elution ion-pairing HPLC with uv detection at 260 nm and with a minimal chemical manipulation of cells. Exponentially growing WiDr cells were harvested by centrifugation, rinsed with phosphate-buffered saline, and carefully counted. The pellets were suspended in a known volume of ice-cold water and deproteinized with an equal volume of 6% trichloroacetic acid. The acid cell extracts (corresponding to 2. 5 x 10(6) cells/100 microl) were centrifuged at 13,000g for 10 min at 4 degrees C. The resulting supernatants were stored at -80 degrees C prior to analysis. Aliquots (100 microl) were neutralized with 4.3 microl saturated Na2CO3 solution prior the injection of 40 microl onto the HPLC column (injection speed 250 microl/min). Chromatographic separations were performed using two Symmetry C18 3. 5-microm (2 x 3.9 x 150 mm) columns (Waters), connected in series equipped with a Sentry guard column (3.9 x 20 mm i.d.) filled with the same packing material. The HPLC columns were kept at 30 degrees C. The mobile phase was delivered at a flow rate of 0.5 ml/min, with the following stepwise gradient elution program: % solvent A/solvent B, 100/0 at 0 min --> 100/0 at 1 min --> 36/64 at 5 min --> 31/69 at 90 min --> 31/69 at 105 min --> 0/100 at 106 min --> 0/100 at 120 min; 50/50 MeOH/solvent B from 121 to 130 min; 100% solvent A from 131 to 160 min. Solvent A contained 0.01 M KH2PO4, 0.01 M tetrabutylammonium chloride, and 0.25% MeOH and was adjusted to pH 7. 0 (550 microl 10 N NaOH for 1 liter solvent A). Solvent B consisted of 0.1 M KH2PO4, 0.028 M tetrabutylammonium chloride, and 30% MeOH and was neutralized to pH 7.0 (1.4 ml 10 N NaOH for 1 liter solvent B). Even though dNTPs are minor components of cell extracts, satisfactory regression coefficients were obtained for their calibration curves (r2 > 0.99) established with the addition-calibration methods up to 120 pmol/40-microl injection. The applicability of the method was demonstrated by in vitro studies of the modulation of NTP and dNTP pools in WiDr colon carcinoma cell lines exposed to various pharmacological concentrations of cytostatic drugs (i.e., FMdC, IUdR, gemcitabine). In conclusion, this optimized, simplified, analytical method enables the simultaneous quantitation of NTP and dNTP and may represent a valuable tool for the detection of minute alterations of cellular dNTP/NTP pools induced by anticancer/antiviral drugs and diseases.     

Fast analysis of pravastatin in production media

High throughput methods (high performance liquid chromatography and capillary electrophoresis) were developed to determine pravastatin in production media. The analyses were performed on particle column, monolithic column and silica capillary filled with borate buffer pH 9.3 containing 20 mM SDS. All three methods successfully separate pravastatin from interfering compounds (matrix, mevastatin and 6-epi pravastatin) and runtimes are shorter than 1 min. Solvent consumptions for methods using small particle column, monolith column and MECK were 132, 510 and 1.5 mL h(-1). The most sensitive was the method using particle column (LOD was about 10(-5) mg mL(-1)), followed by the system using monolith column (LOD was 2 x 10(-4) mg mL(-1)) and the MECK method (LOD was about 0.02 mg mL(-1)).     

Quantification of CPT13 in rat plasma using LC-MS/MS for a pharmacokinetic study

A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using tandem mass spectrometry detection was initially developed and validated for the analysis of 10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) in rat plasma. Pretreatment of the sample obtained from plasma involved a single protein precipitation step with using acetonitrile containing 0.1% formic acid. An aliquot of 20 μl was injected into a C-18 column. The chromatographic separation was achieved using the mobile phase consisting of acetonitrile:water (35:65) at a flow rate of 1.0 mL/min. The total run time for each sample was 10 min, and camptothecin (CPT, IS) and CPT13 were well separated with retention times of 5.1 min and 5.6 min, respectively. Detection was performed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r2 = 0.9998) over the concentration range of 1-1000 ng/mL, with a LLOQ of 1 ng/mL for CPT13. The inter- and intra-day precision (%R.S.D.) were <2.58% and 6.28%, respectively, and the accuracies (%) were within the range of 97.34-110.67%. CPT13 in rat plasma was stable when stored at -20 °C or 4 °C for three freeze-thaw cycles, The method was employed for the first time during pharmacokinetic studies of CPT13 in rats following a single intravenous dose (0.1 mg/kg) and three different oral doses (50 mg/kg, 30 mg/kg, and 10 mg/kg). This fully validated method was successfully applied to a pharmacokinetic study of CPT13 in rats.     

Determination of estradiol 2- and 16 alpha-hydroxylase activities in rat liver microsomes using high-performance liquid chromatography

We have developed a sensitive and nonradiometric assay of estradiol 2- and 16 alpha-hydroxylase activities using reverse-phase high-performance liquid chromatography with voltametric detector. The 2- and 16 alpha-hydroxylated estrogens produced by the incubation of estradiol with rat liver microsomes were initially separated into the catechol and phenolic fractions using a QAE-Sephadex A-25 borate column. The metabolites were detected in quantities as low as 0.5-1 ng using 3-methoxy-1,3,5(10)-estratriene-2,16 alpha,17 beta-triol or 4-hydroxyestrone 17-oxime as an internal standard. Apparent Km and Vmax of the 2- and 16 alpha-hydroxylases were 41.9 microM and 1.3 nmol/mg protein/min, and 82 microM and 480 pmol/mg protein/min, respectively.