Absorption, protein binding, pharmacokinetics and excretion of the anti-ischemic and anti-hypertensive arylpiperazine derivative CDRI-93/478 in rats

CDRI-93/478 (1- [4-(4-fluorophenyl) piperazine-1-yl]-3-(2-oxopyrrolidin-1-yl) propane hydrochloride, an arylpiperazine derivative, is a potent anti-ischemic and anti-hypertensive agent and is in advanced stage of preclinical trials. In order to develop CDRI-93/478 into a clinical agent, the absorption, protein binding, pharmacokinetics, and excretion of the compound were investigated in male Sprague-Dawley rats. Oral absorption was evaluated in situ and in vivo, using the portal-venous concentration difference method. The compound showed negligible absorption (ka = 0.01 h(-1)) at pH 2.6. However, the rate of absorption of the compound at pH 7.4 was 0.6 h(-1) and was comparable to that observed in the in vivo study (ka, >0.58 h(-1)) in rats after a single 2 mg/kg oral dose. In vitro and in vivo protein binding studies using the ultrafiltration method showed that the compound was subject to low protein binding (<40%) and was independent of the substrate concentration over a range of 1-16 microg/ml. Pharmacokinetic parameters of the compound were determined after intravenous and oral administration of 0.6, 2 and 8 mg/kg doses using a model independent method. After oral administration, the compound showed the double-peak phenomenon, which could be due to the high water solubility (log P, 1.01 +/- 0.01), regional differences in the gastrointestinal absorption and enterohepatic recirculation effects. The absorption of CDRI-93/478 was rapid and showed a bioavailability of 69.9 +/- 5.1% (mean +/- S. D.) after 2 and 8 mg/kg oral dose. However, the pharmacokinetic parameters of the compound could not be determined after the 0.6 mg/kg oral dose due to insufficient data points. The studies following intravenous and oral administration demonstrated linear pharmacokinetics, low clearance and high volume of distribution over the dose range studied. The excretion studies after the 8 mg/kg oral dose indicated that the compound was not excreted through the feces and the urinary excretion was very low (<2%).     

LC determination of the anti-ulcer agent CDRI-85/92 in rat serum

CDRI-85/92 is a new potent anti-ulcer compound developed by Central Drug Research Institute, Lucknow (India). This compound is in advanced stage of preclinical trials. A High-performance liquid chromatographic (HPLC) method was developed and validated for the analysis of CDRI-85/92 using rat serum. The HPLC analysis, applicable to 0.5-ml volumes of serum, involved protein precipitation of serum samples with acetonitrile (1:3 v/v) followed by centrifugation and separation on a C-18 column and the use of UV detector at the wavelength 250 nm. The method was sensitive with a lowest limit of quantitation (LLOQ) of 1.25 ng ml⁻¹ in rat serum and the recovery was more than 96%. The linearity was satisfactory as indicated by correlation of >0.99, in addition to the visual examination of the calibration curves. The precision and accuracy were acceptable as indicated by relative standard deviation (R.S.D.) ranging from 4.15 to 8.21%, bias values ranging from 2.96 to 11.18%. In-process stability evaluation showed the stability of the compound in processed samples lasted up to 168 h. The method was applied for analysing CDRI-85/92 in rat serum after administration of single oral or iv bolus dose of 20 mg kg⁻¹. The robustness/ruggedness of the HPLC procedure was tested using different HPLC instrumentation and column of different make. The assay was found to be sensitive (limit of quantification was 1.25 ng ml⁻¹), specific (retention time for CDRI-85/92 is 7.5 min), accurate (% bias is <12%), precise (% R.S.D. is <10%), robust (no significant change in peak profile in two HPLC Instruments) and reliable for use in pharmacokinetic or toxicokinetic studies.     

Liquid chromatographic determination of a non-steroidal oral contraceptive CDRI-85/287 in rat serum

A precise and sensitive high performance liquid chromatographic (HPLC) assay method was developed and validated for the quantitation of 2-[4-(2-piperidinoethoxy) phenyl]-3-phenyl-(2H)-1-benzo(b)pyran (compound CDRI-85/287) in rat serum. This method, applicable to 0.5 ml volumes of serum, was validated according to GLP guidelines. It involved double extraction of serum samples with a mixture of hexane and iso-propanol (98:2 v/v) at alkaline pH and the use of UV detection at 332 nm. Linearity, precision and accuracy were acceptable (5–200 ng ml⁻¹). The absolute recovery was more than 75% and the lower limit of quantitation was 5 ng ml⁻¹. Freeze–thaw stability studies up to four cycles showed no apparent differences in the calculated spiked concentrations. However, in-process stability evaluation showed the stability of the processed samples lasted up to 85 h.     

LC/MS determination of the enaminones E139, DM5 and DM27 in rat serum

Enaminones, E139, DM5 and DM27, have been recently recognized as potential anticonvulsant compounds. The molecular masses of these enarminones were proven using ion trap Finnigan mass spectrometer. For conduction of biological studies in animals, a sensitive and selective high-performance liquid chromatography-mass spectrometry (LC/MS) was developed for the determination of the selected enaminones in rat serum. A simple protein precipitation procedure was followed for cleaning up the serum samples before analysis. LC/MS determinations were performed using an APCI probe at 430 degrees C. Positive ions (M+1)(+) were acquired in MS/MS-SRM mode at m/z 308.1 (parent m/z 340.2) for E139 and m/z 262.1 (parent m/z 294.1) for DM5. On the other hand, DM27 and E118 (internal standard) were measured in SIM mode at m/z 236.5 and 222.5, respectively. Quantitation was based on measurement of the peak area ratio of enaminones (E139, DM5, DM27) and E118 as an internal standard. Calibration curves were linear (r>0.9989) over the concentration range 100-1000 ngml(-1) and were free from serum interference. Precision and accuracy studies of control samples showed intra-day and inter-day %RSD <10.1 and % deviation from nominal concentrations (%DEV) from -4.3 to +10.1. Recoveries of E139, DM5 and DM27 from quality control rat serum samples using protein precipitation method were 92.3, 89.4 and 89.6%, respectively. The reported data suggest the utility of this developed method for structural elucidation and for performing pharmacokinetics studies on the selected enaminones in rats.     

LC determination of a sulphur mustard decontaminant CC-2 in rat serum

A high-performance liquid chromatographic (HPLC) method was developed for the analysis of N,N'-Dichlorobis (2,4,6-trichlorophenyl) urea (CC-2), a potent sulphur mustard decontaminant, in rat serum. The HPLC analysis, applicable to 0.5 ml volumes of serum, involved double extraction of serum samples with diethyl ether at alkaline pH followed by separation on a RP-18 column and the use of UV detector at 230 nm. The method was sensitive with a limit of quantitation of 10 ng ml(-1) in rat serum and the recovery was always >90%. Excellent linear relationships (r>0.99) were obtained between the measured and added concentration ratios of the serum concentrations over a range of 10-200 ng ml(-1). The precision and accuracy were acceptable as indicated by relative standard deviation ranging from 2.47 to 17.49%, bias values ranging from -4.35 to 13.21%. Moreover, CC-2 was found stable in rat serum even after 3 months of storage at -60 degrees C and being subjected to three freeze-thaw cycles. The assay was found to be sensitive, specific, accurate, precise, and reliable for use in pharmacokinetic studies.     

Assay method for quality control and stability studies of a new CVS disorder agent (compound 93/478)

1-[4-(4-fluorophenyl)-piperazine-1-yl]-3-(2-oxopyrrolidin-1-yl)-propane hydrochloride, (I), (CDRI code No. 93/478) is a new potent anti-ischemic and anti-hypertensive agent, being developed at the Central Drug Research Institute (CDRI), Lucknow, India. A sensitive high performance liquid chromatographic assay method has been developed and validated for in process quality control and for stability studies. HPLC separation was achieved on a C(18) Purospher (Merck) column using a gradient of 0.02% tetra-methyl ammonium hydroxide (pH 7.5) and acetonitrile as mobile phase. The eluents were monitored by diode array detector at 240 and 290 nm. The lower limit of detection of I was 0.62 microg/ml, while the lower limit of quantitation was set to be 1.5 microg/ml. The calibration curves were linear in the range 1.5-62 microg/ml. Reproducibility of the method was determined by inter and intra assay variation, which were <10%.     

液-质联用法测定反式白藜芦醇在大鼠和比格犬体内的代谢产物

目的：液相色谱串联多级质谱法测定大鼠和比格犬体内反式白藜芦醇（trans-resveratrol，TR）的代谢产物。方法：大鼠按150mg&#183;kg^-1灌胃给予TR，收集给药后0～12h尿、粪和胆汁样品，比格犬按50mg&#183;kg^-1灌胃给予TR，收集给药后0～12h尿样和粪样。用C18柱固相萃取后进行液-质联用法（LC／MS^n）分析。结果：在服药后的大鼠体内可测到4个Ⅱ相代谢物（2个O-葡萄糖醛酸化和2个硫酸化物），在比格犬体内可测到2个Ⅱ相代谢物（O-硫酸化物）。结论：TR在大鼠和比格犬体内广泛代谢，并存在着种属差异。     

LC and TLC determination of cinnarizine in pharmaceutical preparations and serum

New high performance liquid chromatography (HPLC) and thin layer densitometry (TLC) methods are developed for quantification of cinnarizine in dosage forms in the presence of its photo-degradation products and related substances and in the presence of its metabolites in serum. Mobile phases consisting of benzene-methanol-formic acid (80:17:3) and methanol-acetate buffer of pH 4 (70:30) are satisfactorily used for resolution of cinnarizine from associated substances by TLC and HPLC techniques, respectively. The lower detection limits are 16 and 10 ng microl(-1) of cinnarizine with standard deviations of 1.3 and 1.1% with TLC and HPLC, respectively. The methods are used for assessment of drug purity, stability, bioavailability, bioequivalency and tablet dissolution rate. Four cinnarizine related substances and six drug degradation products are isolated and identified by infrared and mass spectrometry. The results obtained by both techniques are in good agreement and offer the advantages of reproducibility and accuracy.     

Inhibition of rat brain monoamine oxidase activity by cerebral anti-ischemic agent, ifenprodil

Ifenprodil, which is clinically used as a cerebral vasodilator, inhibited rat brain type A (MAO-A) and type B (MAO-B) monoamine oxidase activity. It did not, however, affect rat lung semicarbazide-sensitive amine oxidase. The degree of inhibition of either form of MAO was not changed by 30 min preincubation of the enzyme preparations at 37 degrees C with ifenprodil. Modes of inhibition of MAO-A and MAO-B by ifenprodil were competitive towards oxidation of their respective substrates, 5-hydroxytryptamine and benzylamine, with Ki values of 75 microM for inhibition of MAO-A and 110 microM for inhibition of MAO-B.     

LC determination of oxcarbazepine and its active metabolite in human serum

Twenty-five percent of epileptic patients present refractory seizures to current frontline antiepileptic drugs, needing new treatments and leading to the introduction of several new AEDs, among which is oxcarbazepine (Trileptal). This 10-ketoanalogue of carbamazepine seems to be a weaker inducer of cytochrome P450 3A4. However, pharmacokinetic interactions with clinical significance have already been reported, before the marketing of Trileptal in France. The aim of this study was to develop and validate a HPLC method allowing simultaneous dosage of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenyto?n. After plasma defecation by acetonitrile, dosage was obtained by analysis of the supernatants on a C(18) reversed-phase column coupled with UV detection (240 nm). The statistical validation was performed according to the recommendations of a European technical commission. This method seems to provide a quite good selectivity from the psychotropic therapeutics, which is commonly coprescribed with AEDs. Linearity was established for the whole concentration range, whatever the compound. Quantization limits of oxcarbazepine, 10-hydroxycarbamazepine, epoxycarbamazepine, carbamazepine, phenobarbital and phenyto?n are 0.58, 3.5, 2.35, 0.66, 1.02 and 3.13 microg/ml, respectively, and absolute recoveries are 105.15, 84.76, 94.45, 96.52, 98.62 and 95.08%, respectively.     

A sensitive LC assay for the simultaneous determination of centbutindole and its metabolite in rat serum using fluorescence detection.

Centbutindole (+/-) 2-gamma-[(p-fluorobenzoyl)propyl]-1, 2, 3, 4, 6, 7, 12, 12a-octahydro-pyrazino (2', 1': 6, 1) pyrido [3, 4-b] indole (I), is a new neuroleptic agent developed by Central Drug Research Institute, India. In the present study, a high performance liquid chromatography (HPLC) assay method for the simultaneous assay for I and its metabolite (II) in rat serum was developed and validated. The present method requires only 1 ml of serum with detection levels similar to that reported earlier using 4 ml serum. This assay has been found to be more suited for pre-clinical as well as phase IV studies. Linearity was observed between 1.25 and 40 ng/ml for I and 0.625 and 20 ng/ml for II in rat serum. Recoveries were consistent for both the analytes over the concentration ranges studied. Variation in intra-and inter-batch accuracy and precision were within acceptable limits of +/-20% at lowest limit of quantitation, whereas at higher concentrations it was +/-15%. The assay method was employed for the study of the pharmacokinetics and metabolism of I in rats. The parent compound and its metabolites were quantitated in serum and could be monitored up to 24 h post dose.     

A rapid and sensitive LC/MS/MS assay for quantitative determination of digoxin in rat plasma

Digoxin is a cardiac glycoside that is widely used for the treatment of congestive heart failure. To evaluate pharmacokinetics of digoxin in rats, a sensitive LC/MS/MS assay was developed and validated for the determination of digoxin concentration in rat plasma. For detection, a Sciex API3000 LC/MS/MS with atmospheric pressure ionization (API) mass spectrometry turbo ion spray inlet in the positive ion-multiple reaction monitoring mode was used to monitor precursor→product ions of m/z 798.6→651.6 for digoxin and m/z 577.6→433.3 for oleandrin, the internal standard (IS). The standard curve was linear (r²≥0.999) over the digoxin concentration range of 0.1–100 ng/ml in plasma for digoxin. The mean predicted concentrations of the quality control samples deviated by <5.8% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8.6% relative standard deviation. At the lower limit of quantitation (LLQ) of 0.1 ng/ml, the mean deviation of predicted concentrations from the nominal value was within 3.7%. The extraction recoveries of digoxin and internal standard were 82.7±3.9 and 105.9±2.3%, respectively. The present method was successfully applied to characterization of pharmacokinetic profiles of digoxin in rats after oral administration.     

Metabolism of CDRI-85/92, a new potent anti-ulcer agent, involving cis-trans conversion

CDRI 85/92, an anti-ulcer drug, is a new proton pump inhibitor, currently in an advanced stage of drug development. To know more about the drug it was our objective to delineate/identify the metabolic pathway as well as the enzymes responsible for the formation of metabolites. Metabolism of CDRI-85/92 (cis-5-styryl-2-oxazolidinone-4-carboxylic acid) was investigated in rat liver cellular fractions (S9, microsomes and cytosol) using reverse-phase HPLC and mass spectrometry techniques. Two major metabolites were produced by rat liver S9 fractions and reducing factor generating system from either untreated rats or phenobarbitone (PB)-pretreated rats. Incubation of CDRI-85/92 with postmitochondrial fraction (S9) for 24 h resulted in a cis to trans conversion (metabolite M2). Further cis-trans metabolizing capacity was measured separately in the cytosolic and microsomal fractions. Incubation with the cytosolic fraction resulted in an increased rate of cis-trans conversion, while the microsomal fraction showed no cis to trans conversion, thereby restricting the cis to trans conversion to Phase II enzymes, which are mainly located in the cytosol. Studies with PB-pretreated rat liver S9 fractions resulted in an increased rate of cis to trans conversion. Another metabolite was also present (M1) which was identified as an oxygenated metabolite by mass spectrometry. The major urinary metabolite from CDRI-85/92-treated Sprague-Dawley rats (20 mg/kg p.o.) was identified as M2. Studies using sulfobromophthalein and N-ethylmaleimide, as specific inhibitors of GST, showed a complete absence of metabolism, thus indicating the involvement of GST in the metabolism of CDRI-85/92. This study will be helpful in providing clues about factors influencing the bioavailability of CDRI-85/92 as well as drug-drug interactions.     

Quantitative determination of pravastatin and its biotransformation products in human serum by turbo ion spray LC/MS/MS

A sensitive, specific, accurate and reproducible analytical method was developed and validated to quantify pravastatin (Prav), pravastatin-d5 (Prav-d5), SQ-31906, SQ-31906-d5, and pravastatin lactone (Prav-Lac) in human serum samples. Serum samples (0.5 ml) were acidified and extracted by a solid-phase extraction procedure to isolate all five analytes from human serum. Sample extracts were reconstituted and analyzed by turbo ion spray liquid chromatography/tandem mass spectrometry (LC/MS/MS) in the positive ion mode. The total run time was 9 min between injections. The assay demonstrated a lower limit of quantitation (LLQ) of 0.5 ng/ml for all five analytes. The calibration curves were linear from 0.5 ng ml to 100 ng/ml for all five analytes. The coefficients of determination of all calibration curves were > or = 0.999. Precision and accuracy quality control (QC) samples were prepared at concentrations of 2, 30, 80, and 500 ng/ml for all analytes. The intra-assay and inter-assay precision calculated from QC samples were within 8%, for all analytes. The inter-assay accuracy calculated from QC samples was within 8% for all analytes. The extraction recoveries were > or = 90% for all analytes. Benchtop stability experiments in an ice-water bath ( < or = 10 degrees C) demonstrated that over time, Prav-Lac hydrolyzes to Prav in serum. Prav, Prav-d5, SQ-31906, and SQ-31906-d5 were stable under these conditions for up to 24 h. Hydrolysis was minimized by buffering the serum to pH 4.5 and maintaining the serum sample in an ice-water bath. All analytes were stable after three freeze/thaw cycles and in reconstitution solution after 1 week at 4 degrees C. Stability of all analytes in human serum was demonstrated after storage at -70 degrees C for 77 days. The benchtop (< or = 10 degrees C) stability of pooled study samples was also investigated and the results were comparable to those obtained from serum QC samples.     

Determination of loperamide in rat plasma and bovine serum albumin by LC

A rapid, isocratic liquid chromatographic (LC) method was developed for the determination of loperamide (Lop) in solutions of bovine serum albumin (BSA) and rat plasma. Prior to LC analysis, BSA solutions or rat plasma samples were treated with metaphosphoric acid to precipitate protein. Supernatant was directly injected onto a C18 reverse phase column and loperamide was monitored by a UV detector set at 195 nm. The concentrations of Lop in both rat plasma and BSA solution samples were determined by comparison with their calibration curves, which were generated from the peak area ratio of Lop to internal standard, clomipramine versus loperamide concentration. The calibration curves were linear in the range 0-3.0 microg ml(-1) of Lop for the BSA solution sample and 0-1.0 microg ml(-1)for the rat plasma sample. Overall recoveries of loperamide added to BSA and rat plasma samples were 101.4 and 95.5%, respectively. The method is simple (no extraction), rapid (22 min separation time), sensitive (the detection limit of loperamide is 50 ng ml(-1) for the BSA solution sample and 100 ng ml(-1) for the rat plasma sample), reproducible (within-day R.S.D. of 2.59-7.11%, among-day R.S.D. of 1.25-5.97%), and suitable for routine analysis of loperamide in rat plasma and BSA solution samples.     

Simultaneous determination of Z-SU5416 and its interconvertible geometric E-isomer in rat plasma by LC/MS/MS

SU5416 is a selective inhibitor of vascular endothelial growth factor (VEGF) receptor, which plays a major role in vascular angiogenesis. SU5416 exists as the thermodynamically stable and pharmacologically active cis isomer (Z-isomer) in the solid state. In light-exposed solutions the unstable trans isomer (E-isomer) is formed. The E-isomer is unstable for synthesis and isolation and the analytical standard of the E-isomer is unavailable. A new, simple, fast and reliable LC/MS/MS method was developed to quantify both isomers simultaneously in rat plasma samples in order to support the study of disposition kinetics of Z- and E-SU5416. This method is sensitive (LOQ = 0.5 ng/ml), reproducible, and has a wide linear range (0.5-2500 ng/ml).     

Application and validation of a LC/fluorescence method for the determination of amoxicillin in sheep serum and tissue cage fluid

A LC method with fluorescence detection after pre-column mercury dichloride derivation was developed and validated for the quantitative determination of amoxicillin in sheep blood serum and tissue cage fluid at levels down to 100 and 200 ng/mL, respectively. Spiked blood serum and tissue cage fluid samples were deproteinized, derivatized with mercury dichloride and extracted prior to reversed phase LC analysis with fluorescence spectrophotometric detection at an excitation wavelength of 355 nm and an emission wavelength of 435 nm. Separation was carried out on a C₁₈ column with a mobile phase consisting of phosphate buffer, octanesulphonate sodium (OCT), and acetronitrile. A regression model using 1/concentration weighting was found the most appropriate for quantification. The intra-day precision for serum was 1.65–8.74% and for tissue cage fluid was 2.48–6.27%. The inter-day precision for serum was 0.39–3.57% and for tissue cage fluid was 0.44–2.54%. The overall precision over 3 days for blood serum using of 108 replicates was 1.70–9.44% and for tissue cage fluid using of 54 replicates was 2.51–6.76%. Studies of amoxicillin stability in blood serum and tissue cage fluid indicated that amoxicillin was stable after 4 weeks storage at −85 °C. The method was successfully applied for the determination of amoxicillin in blood serum and tissue cage fluid samples collected from rams after intravenous administration.     

LC/MS determination of bromazepam, clopenthixol, and reserpine in serum of a non-fatal case of intoxication

Combined liquid chromatography and mass spectrometry (LC/MS) with a moving belt interface can be used as a rapid method for the determination of bromazepam, clopenthixol, and reserpine in serum samples obtained from cases of acute overdoses with combinations of these drugs. Low resolution detection limits are about 100 pg for the three drugs, while in high resolution mode the detection limit for bromazepam is shown to be at least 35 pg. Accurate masses were obtained in a serum sample within 5 ppm using high voltage scanning over a narrow mass range for about 10 ng of bromazepam and clopenthixol, respectively. Chemical deactivation of the belt was shown to effectively reduce memory effects and to improve the desorption characteristics of the belt leading to higher yields of evaporated intact molecules.     

新型LC/LC-UV系统建立血浆中葛根素含量测定方法

目的 建立新型LC/LC-UV系统测定血浆中葛根素浓度的方法,用于葛根素人体或动物的药物动力学研究.方法 LC1色谱柱FRO-XBC18（30 mm×4.6 mm,20μm,ANAX）,流动相为20 mmol·L-1醋酸铵溶液-乙腈=75：25（v/v）,流速为1.0 mL·min-1,中央处理体系温度为40℃,LC2系统中分析色谱柱为AC-XB C18（300mm×4.6 mm,5μm,ANAX）,流动相为20 mmol·L-1醋酸铵溶液（用甲酸调pH到3.8）-乙腈=86：14（v/v）,流速为1.2 mL·min-1,检测波长为248 nm.目标物从LC1转移到LC2,聚焦流速为1.0 mL·min-1,目标物转移窗口为1.66～2.20 min;定量环为200μL,样品用15%三氯醋酸预处理.结果 葛根素在0.020～4.50μg·mL-1与峰面积线性关系良好,平均相关系数为0.999 4,最低定量浓度为0.015μg·mL-1.日内RSD～3.0%（n=6）,日间RSD〈5.0%（n=18）,准确度在100.3%～102.6%.结论 新型LC/LC-UV系统的葛根素测定方法操作步骤少、准确性与精密度良好、自动化程度高,适合于葛根素血药浓度测定及动力学研究.