Enantioselective HPLC Method Research Articles

Enantioselective polar-organic mode high-performance liquid chromatographic separation of lifitegrast on immobilized polysaccharide stationary phase and its application to pH-dependent chiral interconversion studies.

(S)-Lifitegrast (LFT) is the novel integrin antagonist, approved by the Food and drug administration, to treat signs and symptoms of dry eye disease. Synthesis of racemic LFT, preparative and analytical enantiomer separation, and chiral interconversion studies are lacking in the literature. Hence, in our study, synthesis of LFT racemate, chiral preparative purification procedure of enantiomer, and comprehensive analytical advancements are focused on rapid enantioselective separation and pH-dependent chiral interconversion studies. The synthesis of LFT racemate employed 2-amino-3-(3-(methylsulfonyl)phenyl)propanoic acid hydrochloride and 2-(benzofuran-6-carbonyl)-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-6-carbonyl chloride as starting materials. (R)-LFT was isolated from the racemate by preparative chiral HPLC and characterized using Q-TOF, FT-IR, NMR spectroscopy, and chiral HPLC. The purity of (R)-LFT was determined to have an enantiomeric excess of 99.12%. A precise, accurate, rapid HPLC-DAD enantioselective analytical method has been developed on Chiralpak IC [tris(3,5-dichloro phenyl carbamate) immobilized on cellulose] using water and methanol as mobile phase. The chiral interconversion study reveals 0.22% and 0.21% of interconversion of (S)-LFT into (R)-LFT at 80°C in pH7.4 and 9.5 buffers, respectively, on the 24th day. An alternative route to enantioselective synthesis of LFT enantiomers by chromatographic separation is proposed. The validated enantioselective HPLC method will help to test the regular quality control samples.

Enantioselective evaluation of chiral cosmetic preservative chlorphenesin on cytotoxicity, pharmacokinetics and tissue distribution

The chiral chlorphenesin is widely used in cosmetics as preservative in a racemate. In this study, the cytotoxicity of chlorphenesin enantiomers to HepG2 cells was measured by MTT assay. Chlorphenesin enantiomers inhibited HepG2 cells proliferation with significant enantioselectivity, and the cytotoxicity of S-chlorphenesin was apparently higher than that of R-chlorphenesin. In order to further explore the enantioselective differences of chlorphenesin in vivo, an enantioselective HPLC method was developed and validated to study the pharmacokinetics and tissue distribution of chlorphenesin enantiomers in rat. Satisfactory enantioseparation of chlorphenesin was achieved on a homemade MDCPC column with methanol–water (50:50, v/v) as mobile phase at a flow rate of 0.6 mL min−1. Detailed validation parameters, including linearity, accuracy, precision and stability met the requirements of bioanalysis. After a single skin administration of 900 mg kg−1 racemic chlorphenesin to rat, the maximum plasma concentration and area under the concentration–time curve of S-chlorphenesin were noticeably 1.80 and 2.84 times higher than those of R-chlorphenesin, respectively, but the clearance of R-chlorphenesin was 2.41 times higher than its enantiomer. Among tissues, stomach, small intestine and liver were the main targeting organs for distribution of chlorphenesin enantiomers. Noticeably, R-chlorphenesin and S-chlorphenesin presented significantly stereodependent distribution in rat tissues, especially in liver, brain and kidney. These results indicated that chlorphenesin enantiomers have stereoselective differences in cytotoxicity, pharmacokinetics and tissue distribution. This work could provide more beneficial supporting information in evaluating the cosmetic safety issues related to the use of chlorphenesin enantiomers.

Analytical quality by design development of an ecologically acceptable enantioselective HPLC method for timolol maleate enantiomeric purity testing on ovomucoid chiral stationary phase

Official method in Ph. Eur. for evaluation of timolol enantiomeric purity is normal-phase high performance liquid chromatography (NP-HPLC) method. Compared to other HPLC modes, NP is depicted as quite expensive with high consumption of organic solvents which leads to chronic exposure of analysts to toxic and carcinogenic effects. In order to overcome above-mentioned drawbacks, the aim of this study was to develop new method with better eco-friendly features. This was enabled by using protein type Chiral Stationary Phase (CSP) in reversed-phase mode that required up to 10 % (v/v) of organic solvent. Therefore, an enantioselective HPLC method was developed and validated for quantification of (S)-timolol and its chiral impurity, (R)-isomer. Optimized separation conditions on ovomucoid column were set using Analytical Quality by Design (AQbD) approach in method development. Optimization step was performed following the Box-Behnken experimental plan and the influence of three critical method parameters (CMPs) towards enantioseparation of the above-mentioned peak pair was examined. CMPs included variation of acetonitrile content in the mobile phase (5–10 %, v/v), pH value of the aqueous phase (6.0–7.0) and ammonium chloride concentration in the aqueous part of the mobile phase (10−30 mmol L−1). The most relevant critical method attributes (CMAs) in this case were the separation criterion between studied critical pair and retention factor of the second eluting peak, (S)-timolol. Qualitative Design Space (DS) was defined by Monte Carlo simulations providing adequate assurance of method’s qualitative robustness (π = 95 %). The selected working point situated in the middle of the DS was characterized by following combination of CMPs: acetonitrile content in the mobile phase 7 % (v/v), pH value of the aqueous phase 6.8 and concentration of ammonium chloride in aqueous phase 14 mmol L–1. In the next step, the quantitative robustness was tested by Plackett-Burman experimental design. The validation studies confirmed adequacy of the proposed method for its intended purpose. Finally, Analytical Eco-Scale metric tool was applied to confirm that developed method represents excellent green analytical method compared to the official one.

Capillary electrophoresis method for the determination of (R)-dapoxetine, (3S)-3-(dimethylamino)-3-phenyl-1-propanol, (S)-3-amino-3-phenyl-1-propanol and 1-naphthol as impurities of dapoxetine hydrochloride

A capillary electrophoresis method was developed and validated for the determination of the purity of dapoxetine with regard to the related substances (3S)-3-amino-3-phenylpropan-1-ol, (3S)-3-(dimethylamino)-3-phenylpropan-1-ol, 1-naphthol and the enantiomer (R)-dapoxetine. The separation was based on a dual selector system, which was optimized by a fractional factorial resolution V + design followed by a central composite face centered design with star distance 1 and Monte Carlo simulations for defining the design space. The optimized background electrolyte consisted of a 50 mM sodium phosphate buffer, pH 6.3, containing 45 mg/mL sulfated γ-cyclodextrin and 40.2 mg/mL 2,6-dimethyl-β-cyclodextrin. Separations were carried out in a 23.5/32 cm, 50 μm fused-silica capillary employing a separation voltage of 9 kV at 15 °C. Following robustness testing using a Plackett-Burman design the method was validated according to the International Council on Harmonization guideline Q2(R1) in the range of 0.05–1.0% relative to the dapoxetine concentration. The method was applied to the analysis of drug substance and a commercial tablet. Data regarding the enantiomeric purity of dapoxetine obtained by the capillary electrophoresis assay were comparable to the data obtained by an enantioselective HPLC method.

Enantiomeric Determination of Omeprazole and Esomeprazole by a Developed and Validated Chiral HPLC Method and Stability Studies by Microthermal Analysis

A rapid, accurate, precise, stability indicating and enantioselective chiral HPLC method was developed and validated for the quantitative (S)- and (R)- omeprazole in omeprazole formulations along with determination of enantiomeric purity of (S)- omeprazole in esomeprazole formulations according to the guidelines of the United States of Pharmacopeia (USP) and International Conference on Harmonization (ICH). The chromatographic separation was achieved with n-hexane/ 2-propanol/ acetic acid/ triethylamine (100 : 20 : 0.2 : 0.1, v/v) at a flow rate of 1.2 ml/min on Chiralcel OD-H and detected at 300 nm. The method showed good linearity, high sensitivity with detection limit (LOD) of 0.71 and 1.16 μg/ml and quantitation limit (LOQ) of 2.16 and 3.51 μg/ml for (S)- and (R)- omeprazole, respectively. The average percentage of recovery was found to be 100.85% to 101.36% for (S)- and 99.81% to 101.62% for (R)- omeprazole. The average percentage of relative standard deviation (% RSD) for intra- and inter- day precision were found to be 0.05% and 0.19% for (S)- and 0.03% and 0.13% for (R)- omeprazole, respectively. Stability study was performed under stress conditions. Microthermal analysis of omeprazole was also performed by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM) to assess the physical and chemical behavior of the drug. The method was successfully applied to the quantitation of (S)- and (R)- omeprazole for omeprazole and as well as determination of (S)- omeprazole purity for esomeprazole formulations.Dhaka Univ. J. Pharm. Sci. 16(2): 221-233, 2017 (December)

Response surface methodology for the determination of the design space of enantiomeric separations on cinchona-based zwitterionic chiral stationary phases by high performance liquid chromatography

Quality-by-Design approach for enantioselective HPLC method development surpasses Quality-by-Testing in offering the optimal separation conditions with the least number of experiments and in its ability to describe the method's Design Space visually which helps to determine enantiorecognition to a significant extent. Although some schemes exist for enantiomeric separations on Cinchona-based zwitterionic stationary phases, the exact design space and the weights by which each of the chromatographic parameters influences the separation have not yet been statistically studied. In the current work, a screening design followed by a Response Surface Methodology optimization design were adopted for enantioseparation optimization of 3 model drugs namely the acidic Fmoc leucine, the amphoteric tryptophan and the basic salbutamol. The screening design proved that the acid/base additives are of utmost importance for the 3 chiral drugs, and that among 3 different pairs of acids and bases, acetic acid and diethylamine is the couple able to provide acceptable resolution at variable conditions. Visualization of the response surface of the retention factor, separation factor and resolution helped describe accurately the magnitude by which each chromatographic factor (% MeOH, concentration and ratio of acid base modifiers) affects the separation while interacting with other parameters. The global optima compromising highest enantioresolution with the least run time for the 3 chiral model drugs varied extremely, where it was best to set low % methanol with equal ratio of acid-base modifiers for the acidic drug, very high % methanol and 10-fold higher concentration of the acid for the amphoteric drug while 20 folds of the base modifier with moderate %methanol were needed for the basic drug. Considering the selected drugs as models for many series of structurally related compounds, the design space defined and the optimum conditions computed are the key for method development on cinchona-based chiral stationary phases.

A Validated Enantioselective HPLC Method for Assay of S-Amlodipine Using Crown Ether as a Chiral Stationary Phase

A Validated Enantioselective HPLC Method for Assay of S-Amlodipine Using Crown Ether as a Chiral Stationary Phase

Enantioselective Disposition of Venlafaxine and its Metabolite O-Desmethylvenlafaxine in Rats

Aim: The study was conducted to understand enantioselective pharmacokinetic of venlafaxine (VEN) and o-desmethylvenlafaxine (ODV) using enantioselective HPLC method. The R-enantiomer of VEN is a potent inhibitor of both serotonin and noradrenaline reuptake, whereas the S-enantiomer is more selective in inhibiting serotonin reuptake. Enantiomers of ODV also inhibit noradrenaline and serotonin reuptake. Materials & methods: Study was carried out in wistar rats after oral administration of 120 mg/kg dose of racemic VEN. Results: Plasma concentrations of S (+) VEN were found to be higher than those of R (-) VEN. Plasma concentrations of S (+) ODV were found to be higher than those of R (-) ODV. Conclusion: A significant difference in most of the pharmacokinetic parameters was observed between the enantiomers of VEN in rat plasma.

In-line coupling of an achiral-chiral column to investigate the enantioselective in vitro metabolism of the pesticide Fenamiphos by human liver microsomes

The distinct activity and toxicity of enantiomers has increased concern about the use of chiral pesticides. The chiral pesticide Fenamiphos (FS) is employed as a racemic mixture to control nematode pests. Although recent studies revealed that FS enantiomers possess different toxicity, the toxicokinetics and liver metabolism of these enantiomers in humans remain unclear. This study characterizes the in vitro metabolism of rac-FS, (+)-FS, and (−)-FS by human liver microsomes and predicts some toxicokinetic parameters. First, a new enantioselective HPLC method was developed to analyze FS and its metabolites [fenamiphos sulfoxide (FSO) and fenamiphos sulfone (FSO2)]. Chiral separation of the stereoisomers was accomplished in an in-line coupled achiral-chiral column (Lichrosorb Si60 − Chiralpak AS-H); hexane: ethanol: methanol (85:12:3, v/v/v) was used as mobile phase at a flow rate of 1.2mLmin−1. Then, the HPLC method was fully validated. All the evaluated parameters agreed with the European Medicines Agency guideline. Finally, the enantioselective kinetic parameters were determined for CYP450 enzymes. The predicted toxicokinetic parameters showed that the liver exclusively eliminated FS without stereoselectivity.

Development and validation of a chiral liquid chromatography method for the determination of MP 3950 enantiomers, a high selective 5-HT4 receptor agonist, in rat plasma and its application to stereoselective pharmacokinetic study.

MP 3950 is an active metabolite of mosapride which exhibits high 5-HT4 receptor agonistic effect. The present paper describes an enantioselective chiral HPLC method for separation and quantification of MP 3950 enantiomers in rat plasma. The plasma samples were prepared by liquid-liquid extraction with ethyl acetate and the baseline chromatographic separation was achieved on a Chiralcel OJ-H column with a mobile phase consisting n-hexane/ethanol/methanol/diethylamine (85:5:10:0.4, v/v/v/v) at the flow rate of 1.0mL/min. The ultraviolet detection was performed at 276nm. The calibration curve was linear in the range from 0.100 to 5.00μg/mL with the lower limit of quantification of 0.100μg/mL for each enantiomer. The intra- and inter-day precisions were not more than 14.7%. The accuracy was from -6.4% to 14.0%. The validated method was successfully applied to chiral inversion and stereoselective pharmacokinetic study in rats after oral administration of MP 3950 racemate. The results indicated that no stereochemical inversion was occurred in rats. And the plasma concentrations and area under plasma concentration-time curve of (S)-MP 3950 were all significantly higher than those of (R)-MP 3950 in both male and female rats, which indicated that the disposition of MP 3950 in rats might be stereoselective.

A Validated Enantioselective HPLC Method for Determination of Ibuprofen Enantiomers in Bulk and Tablet Dosage Form.

A new chiral reversed-phase (RP)-HPLC method with UV detection was developed. Enantioselective resolution of ibuprofen (IBP) was achieved using (3R,4S)-4-(3,5-dinitrobenzamido)-3-(3-(trioxysilyl)- propyl)-1,2,3,4-tetrahydro-phenanthrene [(R,R)-Whelk-O2] chiral stationary phase (4.6 mm id × 250 mm, 10 μm) with a mobile phase composed of ethanol-water (30 + 70, v/v) containing 100 mM ammonium acetate at a flow rate of 1.3 mL/min using diode array detector at λ 220 nm. Calibration curves were linear over the concentration range of 20-180 μg/mL for both IBP enantiomers. Mean % recoveries ±SD of 99.74 ± 1.73 and 99.60 ± 0.93 were obtained for dexibuprofen (dex-IBP) and levoibuprofen (levo-IBP), respectively. Intra- and interday precision calculated as RSD, % were not more than 1.66% for dex-IBP and 1.93% for levo-IBP. The detection limits were 2.09 and 2.06 μg/mL for dex-IBP and levo-IBP, respectively. The method was successfully applied for the determination of dex-IBP in tablet dosage form.

Direct separation of the enantiomers of oxaliplatin on a cellulose-based chiral stationary phase in hydrophilic interaction liquid chromatography mode

(R,R)-oxaliplatin is an anticancer enantiopure active pharmaceutical ingredient. Little attention has been devoted to the analysis of its enantiomeric composition. The enantioselective HPLC method reported in the current Pharmacopoeias shows clear disadvantages with regard to the low resolution and long elution times. In this work, it has been proven the applicability of a last generation polysaccharide-based chiral stationary phase (CSP), i.e. the Chiralpak IC-3, in the enantioseparation of oxaliplatin. Experimental results demonstrated the benefits arising from the development of enantioselective hydrophilic interaction liquid chromatography (HILIC) based strategies. A baseline separation with resolution factor of 5.8 was achieved using a 100mm×4.6mm I.D. IC-3 column set at the temperature of 40°C and a mobile phase consisting of acetonitrile–water 100:5 mixture. At a flow rate of 1mLmin−1 the separation was completed within 8min. The optimized method was proven to be sensitive with LOD and LOQ of the enantiomeric impurity of 0.07 and 0.21μgmL−1, respectively.

Determination of dextromethorphan and levomethorphan in seized heroin samples by enantioselective HPLC and electronic CD

A new enantioselective HPLC method was developed for the resolution and determination of the enantiomers of methorphan, dextromethorphan (DXM) and levomethorphan (LVM), on a Chiralcel® OJ column (250mm×4.6mm I.D.). The resolution of DXM and LVM was obtained using a mobile phase consisting of (n-hexane)-(2-propanol)-diethylamine (70:30:0.1, v/v/v) at a flow rate of 0.5mLmin−1. The enantioselective method was found to be selective (α=1.92) and sensitive (LOD=2.8μgmL−1 for both DXM and LVM). The method was coupled with electronic circular dichroism (CD), allowing the determination of the elution order on the basis of the sign of CD signals of the single enantiomers at 285nm (positive for DXM, negative for LVM).Under the optimized conditions, the validated method was applied to the identification and quantitation of the enantiomers of methorphan in samples of different sources of illicit drugs of abuse (heroin). DXM was found in eight seized samples of street heroin; two of these samples, for which the DXM content was found to be over 5% (w/w) and exceeding a 10% (w/w) ratio with respect to diacetylmorphine, were the cause of two deaths for overdose due to acute narcotism.

Altered brain concentrations of citalopram and escitalopram in P-glycoprotein deficient mice after acute and chronic treatment

According to both in vitro and in vivo data P-glycoprotein (P-gp) may restrict the uptake of several antidepressants into the brain, thus contributing to the poor success rate of current antidepressant therapies. The therapeutic activity of citalopram resides in the S-enantiomer, whereas the R-enantiomer is practically devoid of serotonin reuptake potency. To date, no in vivo data are available that address whether the enantiomers of citalopram and its metabolites are substrates of P-gp. P-gp knockout (abcb1ab (−/−)) and wild-type (abcb1ab (+/+)) mice underwent acute (single-dose) and chronic (two daily doses for 10 days) treatment with citalopram (10mg/kg) or escitalopram (5mg/kg) Serum and brain samples were collected 1–6h after the first or last i.p. injection for subsequent drug analysis by an enantioselective HPLC method. In brain, 3-fold higher concentrations of S- and R-citalopram, and its metabolites, were found in abcb1ab (−/−) mice than in abcb1ab (+/+) mice after both acute and chronic citalopram treatments. After escitalopram treatment, the S-citalopram brain concentration was 3–5 times higher in the knockout mice than in controls. The results provide novel evidence that the enantiomers of citalopram are substrates of P-gp. Possible clinical and toxicological implications of this finding need to be further elucidated.

Enantioselective biodegradation of pharmaceuticals, alprenolol and propranolol, by an activated sludge inoculum

Biodegradation of chiral pharmaceuticals in the environment can be enantioselective. Thus quantification of enantiomeric fractions during the biodegradation process is crucial for assessing the fate of chiral pollutants. This work presents the biodegradation of alprenolol and propranolol using an activated sludge inoculum, monitored by a validated enantioselective HPLC method with fluorescence detection. The enantioseparation was optimized using a vancomycin-based chiral stationary phase under polar ionic mode. The method was validated using a minimal salts medium inoculated with activated sludge as matrix. The method was selective and linear in the range of 10–800ng/ml, with a R2>0.99. The accuracy ranged from 85.0 percent to 103 percent, the recovery ranged from 79.9 percent to 103 percent, and the precision measured by the relative standard deviation (RSD) was <7.18 percent for intra-batch and <5.39 percent for inter-batch assays. The limits of quantification and detection for all enantiomers were 10ng/ml and 2.5ng/ml, respectively. The method was successfully applied to follow the biodegradation of the target pharmaceuticals using an activated sludge inoculum during a fifteen days assay. The results indicated slightly higher biodegradation rates for the S-enantiomeric forms of both beta-blockers. The presence of another carbon source maintained the enantioselective degradation pattern while enhancing biodegradation extent up to fourteen percent.

Enantioselective separation and determination of citalopram enantiomers in pharmaceutical dosage form and bulk drug using experimental design approach on Chiralcel® OC as a chiral stationary phase

Summary An enantioselective HPLC method was developed and validated for the separation and the estimation of citalopram (CIT) enantiomers in bulk drug and pharmaceutical preparations. The method was validated for its linearity (correlation coefficient = 0.9994 and 0.996 for S-(+)-enantiomer and R-(−)-enantiomer, respectively), accuracy, robustness, and intermediate precision. Experimental design was applied during intermediate precision (full factorial 23 design) and robustness testing (Box Benken as a factorial design), for robustness test three factors were considered: percentage of organic modifier, flow rate, and temperature. The separation was achieved on cellulose tris(phenylcarbamate) known as Chiralcel® OC (25 cm, 4.6 mm i.d.) derivatized cellulose (phenyl carbamate), with UV detection at 245 nm using n-hexane-isopropanoldiethylamine (85:15:0.2, υ/υ/υ) as mobile phase at flow rate 0.7 mL min−1. A decrease in the flow rate results in decreasing the selectivity factor (α), while varying the percenta...

Enantioselective separation of the carfentrazone‐ethyl enantiomers in soil, water and wheat by HPLC

A simple enantioselective HPLC method was developed for measuring carfentrazone-ethyl enantiomers. The separation and determination was accomplished on an amylose tris[(S)-alpha-methylbenzylcarbamate] (Chiralpak AS) column using n-hexane/ethanol (98:2, v/v) as mobile phase at a flow rate of 1.0 mL/min with UV detection at 248 nm. The effects of mobile-phase composition and column temperature on the enantioseparation were discussed. The accuracy, precision, linearity, LODs, and LOQ of the method were also investigated. LOD was 0.001 mg/kg in water, 0.015 mg/kg in soil and wheat, with an LOQ of 0.0025 mg/kg in water and 0.05 mg/kg in soil and wheat for each enantiomer of carfentrazone-ethyl. SPE was used for the enrichment and cleanup of soil, water, and wheat samples. Recoveries for two enantiomers were 88.4-106.7% with RSD(r) of 4.2-9.8% at 0.1, 0.5, and 1 mg/kg levels from soil, 85.8-99.5% with the RSD(r) of 4.4-9.6% at 0.005, 0.025, and 0.05 mg/kg levels from water, and from wheat the recoveries were 86.3-91.3% with RSD(r) below 5.0% at 0.2, 0.5, and 1 mg/kg levels. This method could be used to identify and quantify the carfentrazone-ethyl enantiomers in food and environment.

Simultaneous determination of risperidone and 9-hydroxyrisperidone enantiomers in human blood plasma by liquid chromatography with electrochemical detection

Risperidone is a commonly prescribed antipsychotic drug. An enantioselective HPLC method with electrochemical detection was developed and validated for simultaneous determination of plasma concentrations of risperidone and its active metabolites, 9-hydroxyrisperidone enantiomers. Following solid phase extraction of 1.0 mL blood plasma, a baseline separation of the analytes was achieved on an AGP (α 1 acid glycoprotein) column using isocratic mobile phase consisting of methanol–phosphate buffer (pH 6.2; 0.1 M) (15:85, v/v). Total analysis run time was 11 min. For the detection of the analytes analytical cell potentials were set at 500, 650, 950, and 950 mV. The method linearity was attained in the range from 1.0 to 100 ng/mL for risperidone and both 9-hydroxyrisperidone enantiomers. The limit of detection was 0.5 ng/mL for all three analytes. The method was precise and accurate and was successfully applied in a clinical study investigating the stereoselectivity of risperidone 9-hydroxylation.

Influence of blood loss on the pharmacokinetics of citalopram

Extended blood loss results in several compensatory physiological mechanisms, including transfer of extravascular fluid into the blood circulation. If drugs are present in the body, this fluid exchange may imply that blood drug concentrations found in a trauma victim may differ from the concentrations present at the time of the trauma. To address this issue, an animal model was used to investigate the influence of blood loss on pre-existing levels of the antidepressant drug citalopram and its demethylated metabolites. Rats were administered citalopram either acutely (40 mg/kg, orally) or chronically (20 mg/kg daily, subcutaneously) for 6 days using osmotic pumps. In the experimental rats, blood loss was accomplished by withdrawing 0.8 mL blood at 10 min intervals during 70 min. In the control rats, blood was withdrawn at 0 and 70 min only. Blood, brain and lung drug concentrations were analyzed with an enantioselective HPLC method. In the chronically treated rats, the ratios between final and initial citalopram concentrations were 1.08 ± 0.15 and 1.01 ± 0.09 in the experimental rats and controls, respectively, indicating no major effect of blood loss. In contrast, acute oral administration resulted in increased ratios in the exsanguinated rats as compared to controls (1.84 ± 0.50 versus 0.73 ± 0.07; p = 0.0495). In conclusion, the observation of increased blood drug levels in the acute oral rats indicates that absorption of fluid from the gastrointestinal tract may be important in the intravascular refill. Further, in the interpretation of post-mortem blood levels of drugs, these physiological mechanisms should be taken into account.

Development and validation of HPLC methods for the analysis of phenethylamine and indoloquinazoline alkaloids inEvodiaspecies

The aim of this study was to evaluate the chromatographic performance of a PEG stationary phase, in comparison with those of C18 columns, for the HPLC analysis of phenethylamine ((+/-)-synephrine) and indoloquinazoline (rutaecarpine and evodiamine) alkaloids in methanolic extracts of fruits of Evodia rutaecarpa (Juss.) Benth. and E. rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (i.e., E. officinalis Dode) (Rutaceae family). The method was validated and showed good linearity, precision, accuracy, sensitivity, and specificity. The highest content of both phenethylamine and indoloquinazoline alkaloids was found in methanolic fruit extracts of E. rutaecarpa, and it was closely related to the degree of maturity. E. officinalis fruits displayed low amounts of both types of alkaloids. Furthermore, an enantioselective HPLC method for the enantioseparation of (+/-)-synephrine from Evodia fruits was applied, by using a protein-based chiral stationary phase with cellobiohydrolase (CBH) as the chiral selector (Chiral-CBH). Isolation of synephrine from Evodia aqueous fruit extracts was carried out by strong cation-exchange SPE. The results of the application of the method to the analysis of Evodia samples showed that (-)-synephrine was the main component while (+)-synephrine was present in low concentration.