Determination of 2-hydroxyflutamide in human plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

Flutamide is a potent antiandrogen used for the treatment of prostatic cancer. Flutamide undergoes extensive first-pass metabolism to the pharmacologically active metabolite 2-hydroxyflutamide. A simple, sensitive, precise, accurate and specific HPLC method, using carbamazepine as the internal standard, for the determination of 2-hydroxyflutamide in human plasma was developed and validated. After addition of the internal standard, the analytes were isolated from human plasma by liquid–liquid extraction. The method was linear in the 25 to 1000 ng/ml concentration range (r>0.999). Recovery for 2-hydroxyflutamide was greater than 91.4% and for internal standard was 93.6%. The limit of quantitation was 25 ng/ml. Inter-batch precision, expressed as the relative standard deviation (RSD), ranged from 4.3 to 7.9%, and accuracy was better than 93.9%. Analysis of 2-hydroxyflutamide concentrations in plasma samples from 16 healthy volunteers following oral administration of 250 mg of flutamide provided the following pharmacokinetic data (mean±SD): C_max, 776±400 ng/ml; AUC_0–∞, 5368±2689 ng h/ml; AUC_0–t, 5005±2605 ng h/ml; T_max, 2.6±1.6 h; elimination half-life, 5.2±2.0 h.     

Development of a high-performance liquid chromatographic method for the quantification of chlorpyrifos,pyridostigmine bromide,N,N-diethyl-m-toluamide and their metabolites in rat plasma and urine

A method was developed for the separation and quantification of the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate), its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate) and TCP (3,5,6-trichloro-2-pyridinol), the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide), its metabolite N-methyl-3-hydroxypyridinium bromide, the insect repellent DEET (N,N-diethyl-m-toluamide), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method is based on using solid-phase extraction and high-performance liquid chromatography (HPLC) with reversed-phase C₁₈ column, and gradient UV detection ranging between 210 and 280 nm. The compounds were separated using a gradient of 1–85% acetonitrile in water (pH 3.20) at a flow-rate ranging between 1 and 1.7 ml/min over a period of 15 min. The retention times ranged from 5.4 to 13.2 min. The limits of detection ranged between 20 and 150 ng/ml, while the limits of quantitation were between 150 and 200 ng/ml. Average percentage recovery of five spiked plasma samples was 80.2±7.9, 74.9±8.5, 81.7±6.9, 73.1±7.8, 74.3±8.3, 80.8±6.6, 81.6±7.3 and 81.4±6.5, and from urine 79.4±6.9, 77.8±8.4, 83.3±6.6, 72.8±9.0, 76.3±7.7, 83.4±7.9, 81.6±7.9 and 81.8±6.8 for chlorpyrifos, chlorpyrifos-oxon, TCP, pyridostigmine bromide, N-methyl-3-hydroxypyridinium bromide, DEET, m-toluamide and m-toluic acid, respectively. The relationship between peak areas and concentration was linear over a range between 200 and 2000 ng/ml.     

Stereoselective and simultaneous measurement of cis- and trans-isomers of doxepin and N-desmethyldoxepin in plasma or urine by high-performance liquid chromatography

Doxepin is a tricyclic antidepressant marketed as an irrational mixture of cis- and trans-geometric isomers in the ratio of 15:85. A convenient high-performance liquid chromatographic (HPLC) procedure for simultaneous quantitation of geometric isomers of doxepin and N-desmethyldoxepin in plasma and urine is described. The HPLC procedure employed a normal phase system with a silica column and a mobile phase consisting of hexane-methanol-nonylamine (95:5:0.3, v/v/v), a UV detector and nortriptyline as the internal standard. The liquid-liquid extraction solvent was a mixture of n-pentane-isopropanol (95:5, v/v). The limit of quantitation was 1 ng/ml for each isomer. The calibration curves were linear over the ranges 1–200 ng/ml (plasma) and 1–400 ng/ml (urine). In plasma, the accuracy (mean±S.D.) (97.53±1.67%) and precision (3.89±1.65%) data for trans-doxepin were similar to corresponding values for urine, i.e., 97.10±2.40 and 3.82±1.14%. Accuracy and precision data for trans-N-desmethyldoxepin in plasma were 97.57±2.06 and 4.38±3.24%, and in urine were 97.64±3.32 and 5.26±1.83%, respectively. Stability tests under three different conditions of storage indicated no evidence of degradation. The recovery of doxepin was 61–64% from plasma and 63–68% from urine. The method has been applied to analyses of plasma and urine samples from human volunteers and animals dosed with doxepin.     

Chromatographic method for the determination of diazepam,pyridostigmine bromide,and their metabolites in rat plasma and urine

This study describes a chromatographic method for the determination of diazepam, an anxiolytic drug that is also used as an antidote against nerve agent seizures, its metabolites N-desmethyldiazepam, and temazepam, the anti-nerve agent drug pyridostigmine bromide (PB; 3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The compounds were extracted using C₁₈ Sep-Pak Vac 3cc (500 mg) cartridges and separated using isocratic mobile phase of methanol, acetonitrile and water (pH 3.2) (10:40:50) at a flow-rate of 0.5 ml/min in a period of 12 min, and UV detection ranging between 240 and 280 nm. The limits of detection for all analytes ranged between 20 and 50 ng/ml, while limits of quantitation were 100 ng/ml. Average percentage extraction recoveries of five spiked plasma samples were 79.1±7.7, 83.5±6.4, 83.9±5.9, 71.3±6.0 and 77.7±5.6, and from urine 79.4±7.9, 83.1±6.9, 73.6±7.7, 74.3±7.1 and 77.6±5.9 for diazepam, N-desmethyldiazepam, temazepam, pyridostigmine bromide, and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear over the range between 100 and 1000 ng/ml. This method was applied to determine the above analytes following a single oral administration in rats as a tool to study the pharmacokinetic profile of each compound, alone and in combination.     

Simultaneous determination of the histamine H1-receptor antagonist ebastine and its two metabolites,carebastine and hydroxyebastine,in human plasma using high-performance liquid chromatography

Ebastine (CAS 90729-43-4) is an antiallergic agent which selectively and potently blocks histamine H₁-receptors in vivo. A simple and sensitive high-performance liquid chromatography (HPLC) method is described for the simultaneous determination of ebastine and its two oxidized metabolites, carebastine (CAS 90729-42-3) and hydroxyebastine (M–OH), in human plasma. After a pretreatment of plasma sample by solid-phase extraction, ebastine and its metabolites were analyzed on an HPLC system with ultraviolet detection at 254 nm. Chromatography was performed on a cyano column (250×4.0 mm I.D.) at 40 °C with the mobile phase of acetonitrile–methanol–0.012 M ammonium acetate buffer (20:30:48, v/v/v) at a flow rate of 1.2 ml/min. Accurate determinations were possible over the concentration range of 3–1000 ng/ml for the three compounds using 1 ml plasma samples. The intra- and inter-day assay accuracy of this method were within 100±15% of nominal values and the precision did not exceed 12.4% of relative standard deviation. The lower limits of quantitation were 3 ng/ml for ebastine and its metabolites in human plasma. This method was satisfactorily applied to the determination of ebastine and its two oxidized metabolites in human plasma after oral administration of ebastine.     

High-performance liquid chromatographic assay of (±)-ethopropazine and its enantiomers in rat plasma

Two high-performance liquid chromatographic (HPLC) methods are described for determination of (±)-ethopropazine (ET) in rat plasma. After deproteination and liquid–liquid extraction, assay of (±)-ET was performed using either a C₁₈ column (non-stereospecific assay) or an (α-R-naphthyl)ethylurea column (stereospecific assay). The UV detection was at 250 nm. Mean recovery was >85%. Both assays demonstrated excellent linear relationships between peak height ratios and plasma concentrations; quantitation limits were ≤25 ng/ml, based on 100 μl rat plasma. Accuracy and precision were <17% with both methods. Both methods were applied successfully to the measurement of ET plasma concentrations in rats given the drug intravenously.     

High-performance liquid chromatography with fluorometric detection for monitoring of etoposide and its cis-isomer in plasma and leukaemic cells

The podophyllotoxin derivative etoposide, extensively used in anticancer therapy, is highly protein-bound (95%) in plasma. It is a chiral drug and only the trans-isomer is pharmacologically active. Isomerisation to the inactive cis-lactone occurs in plasma. The cis-lacrone is often present in ultrafiltrates of plasma from patients treated with etoposide, therefore it is important to separate the isomers when free etoposide concentrations are assayed. There is reason to believe that free and cellular concentrations are more important for the effect of etoposide therapy than total plasma concentrations. A high-performance liquid chromatographic (HPLC) method for quantification of etoposide and its cis-isomer in plasma, total and non-protein-bound concentrations, and in leukaemic cells is described. After addition of teniposide as internal standard the drugs were extracted with chloroform. Etoposide, its cis-isomer, teniposide and endogenous substances were separated isocratically on a Spherisorb phenyl reversed-phase column. Detection was performed fluorometrically, λ_ex/em = 230/330 nm. Non-protein-bound concentrations were determined after ultrafiltration. The detection limit for etoposide was 10 ng/ml plasma, 25 ng/ml ultrafiltrate and 10 ng/50 · 10⁶ cells. The sensitivity of the assay for the cis-lactone was twice as high due to higher fluorescence. The protein binding of the cis-lactone in plasma from ten healthy blood donors was 54.5±4.8% (mean ± S.D.). Thus, the free fraction was about ten-fold higher than that of the mother compound. The assay is convenient and sensitive enough for the determination of free and cellular fractions of etoposide.     

Prostaglandin F2α concentration in genital tract secretions of dairy bulls

Prostaglandin F_2α (PGF_2α) concentrations in genital tract secretions of conscious dairy bulls were determined by radioimmunoassay procedures and compared with peripheral blood plasma levels. The mean (± SD) PGF_2α concentration of coccygeal venous blood plasma from four bulls was 0.14 ± 0.05 ng/ml. Values for rete testis fluid and seminal plasma were the same, namely 0.17 ± 0.01 ng/ml (n = 5) and 0.17 ± 0.02 ng/ml (n = 4), respectively. However, the PGF_2α level in cauda epididymal plasma was 1.61 ± 0.41 ng/ml, or over 8 to 10 times (P < 0.01) the concentration of any other fluid studied.Added PGF_2α had no effect on the endogenous oxygen consumption of washed cauda epididymal spermatozoa or on the oxidative and glycolytic activities of washed ejaculated spermatozoa in vitro. No evidence was obtained suggesting that the prostaglandin may interact with the stimulatory effect of added testosterone or phosphatidylinositol (PI) on the motility, respiration or glucose uptake of ejaculated spermatozoa.     

High-performance liquid chromatographic determination of spectinomycin in swine,calf and chicken plasma using post-column derivatization

An HPLC method for the determination of spectinomycin in swine, calf and chicken plasma at 0.1 μg/ml or higher is described. The clean-up is based upon ion-pair solid-phase extraction on a High Hydrophobic C₁₈ column treated with sodium dioctyl suflosuccinate. After elution with methanol, spectinomycin is chromatographed on a Spherisorb SCX column using 0.1 M sodium sulphate solution (pH 2.6)-acetonitrile (80:20, v/v) as mobile phase. Fluorescence detection is at an excitation wavelength of 340 nm and an emission wavelength of 460 nm after post-column oxidation with sodium hypochlorite followed by derivatization with o-phthaldialdehyde. Mean recoveries were 99 ± 2% (n = 6), 99 ± 2% (n = 7) and 104 ± 2% (n = 6) for swine, calf and chicken plasma, respectively, at the 0.1 μg/ml level.     

High-performance liquid chromatography using electrochemical detection for the determination of prazosin in biological samples

For the quantitation of prazosin a sensitive high-performance liquid chromatographic (HPLC) method was developed. This HPLC analysis method uses an electrochemical detection technique for the identification and quantitation of prazosin. In this assay the serum samples were deproteinized by using a simple acetonitrile precipitation technique that was followed by n-hexane extraction. Prazosin in the deproteinized serum sample was separated by an isocratic elution with an ODS Hypersil HPLC column (150 × 4.6 mm) using a mobile phase consisting of 0.05 M Na₂HPO₄-acetonitrile (60:40), pH 8.4. Prazosin that was eluted from the column was detected using a Coulochem II electrochemical detector. The precision of this assay method was assessed by performing inter- and intra-assay by spiking prazosin free fetal bovine serum samples with 20 and 40 ng/ml concentrations of prazosin. In the intra-assay the recovery was 95.40±4.82% and 97.80±3.40%, respectively, for 20 and 40 ng/ml concentrations of prazosin that were used to spike the serum samples. This electrochemical detection HPLC assay method could be very useful in monitoring plasma levels of prazosin.     

The relative plasma availabilities of ivermectin in reindeer (<Emphasis Type="Italic">Rangifer tarandus tarandus</Emphasis>) following subcutaneous and two different oral formulation applications

Background

Overwintering (breeding) reindeer (Rangifer tarandus tarandus) are commonly treated with ivermectin against parasitic infestations once yearly in autumn-winter roundups. The only preparations registered to reindeer are those for subcutaneous injection. However, also oral extra-label ivermectin administration is used. Twenty-six, 8-month-old reindeer calves were randomly allocated into three groups. Group 1 (n = 9) received oral ivermectin mixture (Ivomec® vet mixt. 0.8 mg/ml, oral ovine liquid drench formulation), Group 2 (n = 9) oral ivermectin paste (Ivomec® vet 18.7 mg/g equine paste), and Group 3 (n = 8) subcutaneous injection of ivermectin (Ivomec® 10 mg/ml vet inj.), each group at a dose of 200 μg/kg body weight. Blood samples were collected at treatment and at days 1, 2, 3, 6, 9 and 16 post treatment. Plasma concentrations of ivermectin were determined by high-pressure liquid chromatography (HPLC) with fluorescence detection.

Results

The peak plasma concentration (C_max) was reached by 2 days after each treatment. The C_max and Area Under Curve (AUC) differed significantly between the groups: C_max was 30.2 ± 3.9, 14.9 ± 5.7 and 63.1 ± 13.1 ng/ml, and AUC_∞ was 2881 ± 462, 1299 ± 342 and 6718 ± 1620 ng*h/ml for groups 1, 2 and 3, respectively (mean ± standard deviation).

Conclusions

The differences in plasma concentrations of ivermectin are concomitant with earlier observed differences in antiparasitic efficacy, which discounts the use of the equine paste in reindeer in favour of the oral ovine liquid drench formulation, or preferably, the reindeer-registered subcutaneous injection formulation.

     

Metabolism of bucolome in rats: Stability and biliary excretion of bucolome N-glucuronide

Bucolome (BCP) is a non-steroidal anti-inflammatory drug, which is used in the treatment of chronic articular rheumatism. Bucolome N-glucuronide (BCP-NG), a metabolite of BCP, is the first unique N-glucuronide of barbituric acid derivatives. First, the stability of BCP-NG in various pH aqueous solutions was studied. BCP-NG was quite unstable under neutral and acidic conditions, and is easily hydrolyzed to BCP. Based on these characteristics of BCP-NG, a simple, rapid and highly sensitive method for the simultaneous determination of BCP and BCP-NG with phenylbutazone (I.S.) in biological fluids was developed using high-performance liquid chromatography (HPLC). A reversed-phase ODS column was used for the separation of BCP, BCP-NG and I.S. A pharmacokinetic study for BCP and BCP-NG was carried out in male Wistar/ST rats following i.v. administration of BCP at a dose of 10 mg/kg body weight. The slow plasma elimination of BCP with time was shown. A major metabolite of BCP in bile was N-glucuronide. The cumulative amounts of BCP and BCP-NG in the bile over 8 h were approximately 2.4±1.4% and 12.6±2.3% of the dose, respectively. BCP and BCP-NG in the urine were 2.7±0.7% and 3.2±0.3% of the dose. Although BCP had a long half-life (over 8.5 h), the preliminary pharmacokinetic parameters (0–8 h) were determined: t_1/2, 8.52±1.96 h; AUC, 419.9±45.2 μg·h/ml; MRT, 3.29±0.11 h; CL_tot, 5.93±0.54 ml/h; and Vdss, 19.5±1.3 l. These observations are the first pharmacokinetic findings for the N-glucuronide of the barbituric acid derivatives.     

A new mass fragmentographic method for the simultaneous analysis of tryptophan, tryptamine, indole-3-acetic acid, serotonin, and 5-hydroxyindole-3-acetic acid in the same sample of rat brain.

A new method for the concurrent extraction and quantification of tryptophan (Trp), tryptamine (T), indole-3-acetic acid (IAA), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) in samples of rat brain is presented. Homogenization is carried out in 0.1 n HCl containing 1 n KCl and 0.2% NaHSO₃. After centrifugation at 100,000g, the supernatant is percolated through a column of XAD-2 resin, eluted with distilled methanol, and the resulting eluate is evaporated to dryness. The dry residue is then derivatized to yield the pentafluoropropionated (PFP) and methylpentafluoropropionated (Me-PFP) derivatives. Identification and quantification is readily achieved by gas chromatography-mass fragmentographic analysis on a OV-17 or Dexsil 300 column. Endogenous levels in whole rat brain established by this method are IAA, 13,1 ± 2.0 ng/g (n = 6); T, less than 380 pg/g (n = 6); Trp, 4.16 ± 0.23 μg/g (n = 6); 5-HIAA, 442 ± 24 ng/g (n = 6); and 5-HT, 526 ± 81 ng/g (n = 5).     

Simple and sensitive high-performance liquid chromatographic method for the determination of 1,5-benzodiazepine clobazam and its active metabolite N-desmethylclobazam in human serum and urine with application to 1,4-benzodiazepines analysis

A HPLC–UV determination of clobazam and N-desmethylclobazam in human serum and urine is presented. After simple liquid–liquid extraction with dichloromethane the compounds and an internal standard diazepam were separated on a Supelcosil LC-8-DB column at ambient temperature under isocratic conditions using the mobile phase: CH₃CN–water–0.5 M KH₂PO₄–H₃PO₄ (440:540:20:0.4, v/v and 360:580:60:0.4, v/v for serum and urine, respectively). The detection was performed at 228 nm with limits of quantification of 2 ng/ml for serum and 1 ng/ml for urine. Relative standard deviations for intra- and inter-assay precision were found below 8% for both compounds for all the tested concentrations. The described procedure may be easily adapted for several 1,4-benzodiazepines.     

Pharmacokinetics of reboxetine enantiomers in the dog

Reboxetine, (RS)-2-[(RS)-α-(2-ethoxyphenoxy)benzyl]morpholine methanesulphonate, is a racemic compound and consists of a mixture of the (R,R)- and (S,S)-enantiomers. The pharmacokinetics of reboxetine enantiomers were determined in a crossover study in three male beagle dogs. Each animal received the following oral treatments, separated by 1-week washout period: 10 mg/kg reboxetine, 5 mg/kg (R,R)- and 5 mg/kg (S,S)-. Plasma and urinary levels of the reboxetine enantiomers were monitored up to 48 h post-dosing using an enantiospecific HPLC method with fluorimetric detection (LOQ: 1.1 ng/ml in plasma and 5 ng/ml in urine for each enantiomer). After reboxetine administration mean t_max was about 1 h for both enantiomers. C_max and AUC were about 1.5 times higher for the (R,R)- than for the (S,S)-enantiomer, mean values ± SD being 704 ± 330 and 427 ± 175 ng/ml for C_max and 2,876 ± 1,354 and 1,998 ± 848 ng.h/ml for AUC, respectively. No differences between the (R,R)- and (S,S)-enantiomers were observed in t½ (3.9 h). Total recovery of the two enantiomers in urine was similar, the Ae (0–48 h) being 1.3 ± 0.7 and 1.1 ± 0.7% of the enantiomer dose for the (R,R)- and the (S,S)-enantiomers, respectively. No marked differences in the main plasma pharmacokinetic parameters were found for either enantiomer on administration of the single enantiomers or reboxetine. No chiral inversion was observed after administration of the separate enantiomers, as already observed in humans. Chirality 9:303–306, 1997. © 1997 Wiley-Liss, Inc.     

Simultaneous determination of histamine and Nτ-methylhistamine in human plasma and urine by gas chromatography-mass spectrometry

A new and sensitive method is described for the determination of histamine and N^τ-methylhistamine in human plasma and urine by gas chromatography-mass spectrometry. ¹⁵N₂-Labeled histamine and N^τ-[methyl-d₃]methylhistamine were used as internal standards. Histamine and N^τ-methylhistamine were converted to the derivatives N^α-heptafluorobutyryl-N^τ-ethoxycarbonylhistamine and N^α-heptafluorobutyryl-N^τ-methylhistamine, respectively. After these derivatives had been purified on a small column packed with CPG-10, the molecular ions were monitored during selected ion monitoring. Linear standard curves were obtained in the range of 0.5–10 ng/ml for both compounds. The reliability of the histamine analysis was demonstrated by using two different ion pairs, while a comparison with results from two different derivatizations on the same urine sample also established the specificity of the N^τ-methylhistamine analysis. An increase of 1 ng of histamine in the plasma could be precisely determined by the present method. The histamine content of plasma from five normal subjects was determined as 0.83 ÷ 0.37 (S.D.) ng/ml and the N^τ-methylhistamine content in most subjects was below the limits of this measurement. High excretion of histamine was noted in the urine collected in the early morning from a patient with nephritis.     

A radioimmunoassay for deoxycorticosterone in human peripheral plasma using sephadex LH-20 chromatography

An accurate, precise, sensitive and relatively simple radioimmunoassay method for the measurement of deoxyocorticosterone (DOC) in human plasma is described. A hexane pre-extraction Is carried out when the sample contains a high progesterone level. A Sephadex LH-20 column (45 × 0.9 cm, in the system dichloromethane:methanol, 98:2) provides adequate purification before radioimmunoassay. However, it has been observed that progesterone inteference is likely in plasma of pregnant females. A specific antibody was generated against DOC-3-oxime coupled to bovine albumin. The Intra and inter assay precision yielded a coefficient of variation of 12.4% for five samples and 27% for 17 samples. The accuracy was checked by measuring recovery of DOC added to pre-extracted plasma (98.5 ± 12.4 (S.D.)%). The sensitivity (10 pg) and blank values (1.5 ng/100 ml) are satisfactory. The normal plasma DOC level obtained (6.4 ± 4.4 ng/100 ml, n = 14) is in agreement with previously reported values.     

Novel liquid chromatographic assay for the low-level determination of apomorphine in plasma

A novel HPLC assay which is rapid, reproducible and sensitive has been developed for the analysis of apomorphine in plasma. The assay incorporates boldine as an internal standard, and uses solid-phase extraction on C₁₈ mini-columns for sample clean-up and concentration, so enabling quantitation of apomorphine at 500 pg/ml using fluorescence detection (λ_ex 270 nm, λ_em). The HPLC assay comprised a 25 cm-long Techopakk C₁₈ column and a mobile phase of (0.25 M sodium dihydrogen phosphate plus 0.25% heptane sulphonic acid, to pH 3.3 with orthophosphoric acid) containing 30% (v/v) methanol and 0.003% (w/v) EDTA, run at a flow-rate of 1.5 ml/min. Calibration plots prepared in plasma were linear over the range 1–30 ng/ml, (limit of quantitation (LOQ)=490 PG/ML) with R.S.D. of 0.05% and R.E. of 5.0% at the level of 1 ng/ml. Preliminary pharmacokinetic data from two patients given apomorphine by 12 h subcutaneous infusion (patient A dose=35 mg and patient B dose=141 mg) showed apomorphine elimination from plasma to fit a two-compartment model, with initial half-lives of 8.2 and 46.6 min, elimination half-lives of 76.4 and 166.5 min and area under the plasma concentration-time curve (AUC) values of 236 and 405 ng h/ml, respectively.     

Poloxamer-chitosan-based Naringenin nanoformulation used in brain targeting for the treatment of cerebral ischemia

ObjectiveHere, the aim is to improve the bioavailability of Naringenin (NRG) in brain and to establish the highest remedial benefit from a novel anti-ischemic medicine i.e. NRG.MethodsA novel Naringenin-loaded-nanoemulsion (NE)-(in situ)-gel (i.e. thermoresponsive), was formulated with the help of Poloxamer-407 (20.0% w/v). Chitosan (CS, 0.50% w/v) was used to introduce the mucoadhesive property of NE-(in situ)-gel and finally called as NRG-NE-gel + 0.50%CS. A novel UHPLC-ESI-Q-TOF-MS/MS-method was optimized and used for NRG-NE-gel + 0.50%CS to quantify the Pharmacokinetic-(PK)-parameters in plasma as well as brain and to evaluate the cerebral ischemic parameters after MCAO i.e. locomotor activity, grip strength, antioxidant activity, and quantity the infarction volume in neurons with the safety/toxicity of NRG-NE-gel + 0.50%CS after i.n. administration in the rats.ResultsThe mucoadhesive potency and gelling temperature of NRG-NE-gel + 0.50%CS were observed 6245.38 dynes/cm² and 28.3 ± 1.0 °C, respectively. Poloxamer-407 based free micelles size was observed 98.31 ± 1.17 nm with PDI (0.386 ± 0.021). The pH and viscosity of NRG-NE-gel + 0.50%CS were found to be 6.0 ± 0.20 and 2447 ± 24cp (at 35.0 ± 1.0 °C temperature), respectively. An elution time and m/z NRG were observed 1.78 min and 270.97/150.96 with 1.22 min and m/z of 301.01/150.98 for Quercetin (IS) respectively. Inter and intra %precision and %accuracy was validated 1.01–3.37% and 95.10–99.30% with a linear dynamic range (1.00 to 2000.00 ng/ml). AUC_0-24 of plasma & brain were observed 995.60 ± 24.59 and 5600.99 ± 144.92 (ng min/ml g) in the rats after the intranasal (i.n.) administration of NRG-NE-gel + 0.50%CS. No toxicological response were not found in terms of mortalities, any-change morphologically i.e. in the microstructure of brain as well as nasal mucosa tissues, and also not found any visual signs in terms of inflammatory or necrosis.ConclusionIntranasally administered NRG-NE-gel + 0.50%CS enhanced the bioavailability of Naringenin in the brain. In the cerebral ischemic rats, significantly improved the neurobehavioral activity (locomotor & grip strength) followed by antioxidant activity as well as infarction volume. Finally, the toxicity studies carried out and established the safe nature of optimized-NRG-NE-gel + 0.50%CS.     

Solid-phase extraction on Styrosorb cartridges as a sample pretreatment method in the stereoselective analysis of propranolol in human serum

Sample pretreatment using solid-phase extraction (SPE) on cartridges filled with small-particle Styrosorb porous polystyrene-based sorbent has been used in the analysis of propranolol enantiomers in human serum by high-performance liquid chromatography (HPLC) with fluorescent detection. SPE on Sep-Pak C₁₈ cartridges was used as a reference pretreatment method. The propranolol content of the samples was determined by achiral normal-phase HPLC and the enantiomeric ratio of propranolol (S/R) was then determined by chiral HPLC on a column with silica-bonded cellulose-tris(3,5-dimethylphenyl carbamate). Recoveries of propranolol from serum using SPE on Styrosorb and C₁₈ phases were 97±5% and 96±5%, respectively. Detection and quantification limits for propranolol enantiomers were 4 and 7 ng/ml, respectively.