A novel high sensitivity HPLC assay for topiramate, using 4-chloro-7-nitrobenzofurazan as pre-column fluorescence derivatizing agent

A new, sensitive and simple high-performance liquid chromatographic method for analysis of topiramate, an antiepileptic agent, using 4-chloro-7-nitrobenzofurazan as pre-column derivatization agent is described. Following liquid-liquid extraction of topiramate and an internal standard (amlodipine) from human serum, derivatization of the drugs was performed by the labeling agent in the presence of dichloromethane, methanol, acetonitrile and borate buffer (0.05 M; pH 10.6). A mixture of sodium phosphate buffer (0.05 M; pH 2.4): methanol (35:65 v/v) was eluted as mobile phase and chromatographic separation was achieved using a Shimpack CLC-C18 (150 x 4.6 mm) column. In this method the limit of quantification of 0.01 microg/mL was obtained and the procedure was validated over the concentration range of 0.01 to 12.8 microg/mL. No interferences were found from commonly co-administrated antiepileptic drugs including phenytoin, phenobarbital carbamazepine, lamotrigine, zonisamide, primidone, gabapentin, vigabatrin, and ethosuximide. The analysis performance was carried-out in terms of specificity, sensitivity, linearity, precision, accuracy and stability and the method was shown to be accurate, with intra-day and inter-day accuracy from -3.4 to 10% and precise, with intra-day and inter-day precision from 1.1 to 18%.     

Simultaneous HPLC–UV analysis of rufinamide,zonisamide, lamotrigine,oxcarbazepine monohydroxy derivative and felbamate in deproteinized plasma of patients with epilepsy

We present an implementation of a method we previously reported allowing the newer antiepileptic drugs (AEDs) rufinamide (RFN) and zonisamide (ZNS) to be simultaneously determined with lamotrigine (LTG), oxcarbazepine's (OXC) main active metabolite monohydroxycarbamazepine (MHD) and felbamate (FBM) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma samples (250 μL) were deproteinized by 1 mL acetonitrile spiked with citalopram as internal standard (I.S.). HPLC analysis was carried out on a Synergi 4 μm Hydro-RP, 250 mm × 4.6 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5), acetonitrile and methanol (65:26.2:8.8, v/v/v) at an isocratic flow rate of 0.8 mL/min. The UV detector was set at 210 nm. The chromatographic run lasted 19 min. Commonly coprescribed AEDs did not interfere with the assay. Calibration curves were linear for both AEDs over a range of 2–40 μg/mL for RFN and 2–80 μg/mL for ZNS. The limit of quantitation was 2 μg/mL for both analytes and the absolute recovery ranged from 97% to 103% for RFN, ZNS and the I.S. Intra- and interassay precision and accuracy were lower than 10% at all tested concentrations. The present study describes the first simple and validated method for RFN determination in plasma of patients with epilepsy. By grouping different new AEDs in the same assay the method can be advantageous for therapeutic drug monitoring (TDM).     

Stereoselective determination of vigabatrin enantiomers in human plasma by high performance liquid chromatography using UV detection

A rapid and simple high-performance liquid chromatographic method for the determination of the R-(-)- and S-(+)-enantiomers of the antiepileptic drug vigabatrin in human plasma is described. After adding the internal standard (1-aminomethyl-cycloheptyl-acetic acid), plasma samples (200 microL) are deproteinized with acetonitrile and the supernatant is derivatized with 2,4,6 trinitrobenzene sulfonic acid (TNBSA). Separation is achieved on a reversed-phase cellulose-based chiral column (Chiralcel-ODR, 250 mm x 4.6 mm i.d.) using 0.05 M potassium hexafluorophosphate (pH 4.5)/acetonitrile/ethanol (50:40:10 vol/vol/vol) as mobile phase at a flow-rate of 0.9 mL/min. Chromatographic selectivity is improved by concentrating the derivatives on High Performance Extraction Disk Cartridges prior to injection. Detection is at 340 nm. Calibration curves are linear (r(2)> or =0.999) over the range of 0.5-40 microg/mL for each enantiomer, with a limit of quantification of 0.5 microg/mL for both analytes. The assay is suitable for therapeutic drug monitoring and for single-dose pharmacokinetic studies in man.     

Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes

Methadone and buprenorphine are two of the drugs most frequently used for abstinence from illicit opioids and in the treatment of pain. A sensitive and selective high-performance liquid chromatographic method with diode array detection for the simultaneous determination of methadone, buprenorphine and norbuprenorphine has been developed. Separation of the three analytes was obtained by using a reversed-phase column (C8, 250mmx4.6mm i.d., 5microm) and a mobile phase composed of 40% phosphate buffer containing triethylamine, 50% methanol and 10% acetonitrile (final apparent pH 6.0). Loxapine was used as the internal standard. An accurate pre-treatment procedure of biological samples was developed, using solid-phase extraction with C8 cartridges (100mg, 1mL) and needing small amounts of plasma or urine (300microL). The calibration curves were linear over a working range of 10.0-1500.0ng/mL for methadone and of 5.0-500.0ng/mL for buprenorphine and norbuprenorphine in both matrices. The limit of quantitation (LOQ) and the limit of detection (LOD) were 1.0 and 0.4ng/mL for methadone and 0.5 and 0.2ng/mL for both buprenorphine and norbuprenorphine, respectively. The method was successfully applied to the analysis of plasma and urine samples from patients undergoing treatment with these drugs. Precision and accuracy results were satisfactory and no interference from endogenous or exogenous compounds was found. The method is suitable for the simultaneous determination of methadone and buprenorphine in human plasma and urine for therapeutic drug monitoring purposes.     

Simple and validated HPLC-UV analysis of levetiracetam in deproteinized plasma of patients with epilepsy

We present a simple, fast and validated method for the determination of the new generation antiepileptic drug (AED) levetiracetam (LEV) in plasma of patients with epilepsy using high performance liquid chromatography (HPLC) with UV detection. Plasma sample (500 microL) pretreatment was based on simple deproteinization by methanol spiked with the internal standard (I.S.). HPLC analysis was carried out on a Synergi 4-microm Hydro-RP, 150 mm x 4 mm I.D. column. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (50 mM, pH 4.5) and acetonitrile (94:6, v/v) at an isocratic flow rate of 1.5 mL/min. The UV detector was set at 205 nm. Calibration curves were linear (mean correlation coefficient=0.999) over a range of 4-80 microg/mL. The quantitation limit was 2 microg/mL and the absolute recovery was >90% for LEV and the I.S. Both intra and interassay precision and accuracy were lower than 7.5%. The chromatographic run lasted 13 min. The present procedure omitting expensive solid phase or time-consuming liquid-liquid extraction and drying steps is cheaper, faster and simpler than mostly published analytical methods for levetiracetam. Applied to a large population of patients with epilepsy this assay proved very practical in our therapeutic drug monitoring setting (TDM).     

Liquid chromatographic method for the simultaneous determination of cefalexin and trimethoprim in dog plasma and application to the pharmacokinetic studies of a coformulated preparation

A liquid chromatographic method is described for the simultaneous determination of cefalexin and trimethoprim in dog plasma. A simple protein precipitation procedure was adopted for the sample preparation with satisfactory extraction recoveries for both analytes. Chromatographic separation of the analytes was achieved on a C(18) column using a mixture of 2 mol/l formate buffer (pH 3.5), methanol and acetonitrile (22:7:7, v/v/v) containing a 0.002 mol/l sodium dodecyl sulfate as mobile phase and detection was performed at 240 nm. The linearity was obtained over the concentration ranges of 1.0-100.0 microg/ml for cefalexin and 0.5-50.0 microg/ml for trimethoprim. For each level of QC samples including the lower limit of quantification, both inter- and intra-day precisions (R.S.D.) were < or =14.0% for cefalexin and < or =11.4% for trimethoprim, and accuracy (RE) was -1.4% for cefalexin and -3.0% for trimethoprim. The present LC method was successfully applied to the pharmacokinetic studies of coformulated cefalexin dispersible tablets after oral administration to beagle dogs.     

Determination of lamotrigine in small volumes of plasma by high-performance liquid chromatography

Lamotrigine is a broad-spectrum antiepileptic agent. This study describes a simple and sensitive high-performance liquid chromatographic method for the determination of lamotrigine in 50 microl of plasma. Lamotrigine and the internal standard guanabenz were extracted with 1.2 ml of diethyl ether, after the samples alkalinized with 10 microl of sodium hydroxide solution (1N). Chromatographic separation was achieved on a silica column with the mobile phase of acetonitrile-water containing 0.2% phosphoric acid and 0.3% triethylamine (pH 2.7) (84:16, v/v), at a flow-rate of 1 ml/min. The eluant was detected at 225 nm. The retention time was about 6 min for lamotrigine and 7 min for guanabenz. No endogenous substances and concomitant anticonvulsants were found to interfere. Calibration curves were linear from 0.1 to 5 microg/ml. The relative recovery of lamotrigine averaged about 80%. The limit of quantitation was 0.1 microg/ml. The intra- and inter-day precision (expressed as coefficient of variation, CV) was 8.1%, or less, and the accuracy was within 11.5% deviation of the nominal concentration. The method is suitable in pharmacokinetic investigation and monitoring lamotrigine concentration.     

Sensitive high-performance liquid chromatographic quantitation of gabapentin in human serum using liquid-liquid extraction and pre-column derivatization with 9-fluorenylmethyl chloroformate

Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum are based on the same approach, involving o-phthaldialdehyde derivatization of deproteinized serum samples. The present paper however, describes a new, simple and sensitive high-performance liquid chromatographic method for determination of gabapentin in human serum using liquid-liquid extraction and 9-fluorenylmethyl chloroformate (FMOC-Cl) as pre-column labeling agent. The drug and an internal standard (azithromycin) were extracted from serum by salting-out approach using a mixture of dichloromethane-2 propanol (1:1, v/v) as the extracting solvent. The extracted analytes were subjected to derivatization with FMOC-Cl in the presence of phosphate buffer (pH 7). A mobile phase consisting of methanol-0.05 M sodium phosphate buffer (73/27, v/v; pH of 3.9) containing 1 ml/l triethylamine was eluted and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6 mm) column. The standard curve was linear over the range of 0.03-20 microg/ml and limit of quantification was 0.03 microg/ml. The performance of analysis was studied and the validated method showed excellent performance in terms of selectivity, specificity, sensitivity, precision and accuracy. No interferences were found from commonly co-administered antiepileptic agents.     

Determination of epimedin C in rat plasma by reversed-phase high-performance chromatography after oral administration of Herba Epimedii extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of epimedin C in rat plasma and applied to a pharmacokinetic study in rats after administration of Herba Epimedii extract. After addition of carbamazepine as an internal standard plasma samples were extracted with ethyl acetate. HPLC analysis of the extracts was performed on a Hypersil ODS2 analytical column using acetonitrile -0.4% acetic acid (25:75, v/v) as the mobile phase. The UV detector was set at 260 nm. The standard curve was linear over the range 0.05-4.0 microg/mL. The lower limit of quantification was 0.05 microg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of epimedin C in rat plasma after giving the animals Herba Epimedii extract.     

Analysis of risperidone and 9-hydroxyrisperidone in human plasma, urine and saliva by MEPS-LC-UV

Risperidone is currently one of the most frequently prescribed atypical antipsychotic drugs; its main active metabolite 9-hydroxyrisperidone contributes significantly to the therapeutic effects observed. An original analytical method is presented for the simultaneous analysis of risperidone and the metabolite in plasma, urine and saliva by high-performance liquid chromatography coupled to an original sample pre-treatment procedure based on micro-extraction by packed sorbent (MEPS). The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 73% (v/v) acidic phosphate buffer (30 mM, pH 3.0) containing 0.23% triethylamine and 27% (v/v) acetonitrile. The UV detector was set at 238 nm and diphenhydramine was used as the internal standard. The sample pre-treatment by MEPS was carried out on a C8 sorbent. The extraction yields values were higher than 92% for risperidone and 90% for 9-hydroxyrisperidone, with RSD for precision always lower than 7.9% for both analytes. Limit of quantification values in the different matrices were 4 ng/mL or lower for risperidone and 6 ng/mL or lower for the metabolite. The method was successfully applied to plasma, urine and saliva samples from psychotic patients undergoing therapy with risperidone, with satisfactory accuracy results (recovery>89%) and no interference from other drugs. Thus, the method seems to be suitable for the therapeutic drug monitoring of schizophrenic patients using the three different biological matrices plasma, urine and saliva.     

Simultaneous determination of 3,3',4',5,7-pentamethylquercetin and its possible metabolite 3,3',4',7-tetramethylquercetin in dog plasma by liquid chromatography-tandem mass spectrometry and its application to preclinical pharmacokinetic study

A sensitive, simple and rapid ultra fast liquid chromatography (UFLC)-ESI-MS/MS method was established for the simultaneous determination of 3,3',4',5,7-pentamethylquercetin (PMQ) and its possible metabolite 3,3',4',7-tetramethylquercetin (TMQ) in dog plasma using 4',5,7-trimethylapigenin (TMA) as the internal standard. The plasma sample was pretreated with acetonitrile for protein precipitation and the analytes were separated on an Ultimate XB-CN column (5 μm, 2.1 mm × 150 mm) with the mobile phase consisting of acetonitrile and water (2:1, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer under a positive multiple reaction-monitoring mode (MRM). The mass transition ion-pair was followed as m/z 373.1-312.1 for PMQ, 359.1-344.0 for TMQ and 313.1-298.1 for TMA. The validated concentration ranged from 1.272 to 3060 ng/mL for PMQ and from 10.35 to 1725 ng/mL for TMQ. The lower limit of quantifications for PMQ and TMQ were 1.272 ng/mL and 10.35 ng/mL, respectively. The developed-method was successfully applied for the pharmacokinetic study of PMQ and its metabolite TMQ in dogs following a single oral dose.     

Robust isocratic high performance liquid chromatographic method for simultaneous determination of seven antiepileptic drugs including lamotrigine, oxcarbazepine and zonisamide in serum after solid-phase extraction

A rapid, simple and robust method is presented for the simultaneous determination of seven antiepileptic drugs (AEDs), including primidone, phenobarbital, phenytoin, carbamazepine with its two major metabolites carbamazepine-10,11-epoxide and carbamazepine-10,11-(trans)-dihydrodiol and the new AEDs lamotrigine, hydroxycarbazepine (active metabolite of oxcarbazepine) and zonisamide in serum by high performance liquid chromatography (HPLC)-diode array detector (DAD). After solid-phase extraction, separation is achieved on an Alltima 3C18 analytical column using isocratic elution with a mixture of acetonitrile, methanol and phosphate buffer at 45 degrees C. The method is exhaustively validated, including experimental design in combination with statistical evaluation (ANOVA) to study the robustness of chromatography and sample preparation. Commonly co-administered antiepileptic drugs do not interfere with the method. Intra-day precision (RSD<1.9%), linearity, lower limit of quantitation (LOQ<0.065 mg/l) and robustness make the method suitable for daily therapeutic drug monitoring and pharmacokinetic studies.     

Simultaneous determination of amodiaquine and its active metabolite in human blood by ion-pair liquid chromatography-tandem mass spectrometry

A sensitive and selective ion-pair liquid chromatography-tandem mass spectrometric method (IP-LC-MS/MS) for the simultaneous determination of amodiaquine (AQ) and its active metabolite, N-desethylamodiaquine (AQm), in human blood has been developed and validated. Pentafluoropropionic acid (PFPA) was applied as ion-pairing reagent in reversed-phase chromatographic separation. The effects of PFPA concentrations and the volume fraction of acetonitrile in the mobile phase on the retention of analytes were investigated on a Venusil MP-C(18) column, and the mobile phase was finally optimized as acetonitrile:water (23:77, v/v) with 0.0667% PFPA in the aqueous phase. The results proved that PFPA as an ion-pairing reagent could provide desirable chromatographic performance in the IP-LC-MS/MS determination of 4-aminoquinoline compounds. Blood samples were protein precipitated with acetonitrile using hydroxychloroquine (OHCQ) as the internal standard. The detection was carried out in multiple reaction monitoring (MRM) mode via positive atmospheric pressure chemical ionization (APCI) interface. The lower limits of quantification were established at 0.150 and 1.50 ng/mL for AQ and AQm, respectively. The validated IP-LC-MS/MS method was applied to a clinical pharmacokinetic study of AQ and AQm in human blood after an oral administration of 600 mg AQ hydrochloride (45 9mg base).     

A simple and rapid liquid chromatography method for simultaneous determination of zidovudine and nevirapine in plasma

We describe a simple, fast, isocratic, reversed-phase high performance liquid chromatographic method for simultaneous determination of plasma zidovudine and nevirapine with UV detection at 260 nm. The method involves liquid-liquid extraction with ethyl acetate and using 3-isobutyl 1-methyl xanthine as internal standard. The system requires a C(18) column (150 mm x 4.6 mm I.D.) and a mobile phase composed of potassium dihydrogen phosphate (15 mM; pH 7.5) and acetonitrile in the ratio of 80:20 (v/v). The assay was linear from 0.025 to 10.0 microg/ml for zidovudine and 0.05 to 10.0 microg/ml for nevirapine. The intra- and inter-day variations were less than 10% for both the drugs. The method was specific and sensitive enough to allow quantification of zidovudine and nevirapine in concentrations observed clinically. The average recoveries of zidovudine and nevirapine from plasma were 95 and 94%, respectively. The method was applied to a pharmacokinetic study in HIV-infected patients who were receiving antiretroviral treatment with zidovudine and nevirapine containing regimens. The method spans the blood concentration range of clinical interest. Due to its simplicity, the assay can be used for pharmacokinetic studies and therapeutic drug monitoring in patients taking a combination treatment of zidovudine and nevirapine.     

Simultaneous high-performance liquid chromatographic determination of Cedrus deodara active constituents and their pharmacokinetic profile in mice

A specific and sensitive high-performance liquid chromatographic (HPLC) method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of three bioactive constituents of Cedrus deodara namely wikstromol, matairesinol and dibenzylbutyrolactol in mouse plasma. In solid-phase extraction (SPE) these constituents were successfully separated using a C18 column by isocratic elution using acetonitrile:water containing hexanesulphonic acid, 32:68 (v/v). The flow rate was set at 1ml/min and detector wavelength at 225nm. Good linearity (r2>0.999) was observed over the studied range of 0.015-5.0microg/ml for wikstromol and 0.030-5.0microg/ml for matairesinol and dibenzylbutyrolactol. The CV values of intra-day precision for wikstromol, matairesinol and dibenzylbutyrolactol were in between 1.8-6.9, 1.7-4.9 and 1.6-4.2% and values of inter-day precision were in between 10.4-12.2, 9.7-11 and 10-11.2%, respectively. The extraction recoveries at low to high concentration were greater than 98, 83 and 87% for each analyte, respectively. The LOQ for wikstromol was 0.015microg/ml and for both matairesinol and dibenzylbutyrolactol it was 0.030microg/ml. The developed method was used to determine the pharmacokinetics of the three analytes in mice after intraperitoneal administration of CD-3.     

Simultaneous determination of the major active components of tea polyphenols in rat plasma by a simple and specific HPLC assay

A simple and specific HPLC assay for simultaneous determination of two major active components (-) epigallocatechin-3-gallate (EGCG), and (-) epicatechin-3-gallate (ECG) of tea polyphenols (TP) in rat plasma was developed and validated. Following addition of resorcinol as internal standard (IS) the analytes were isolated from rat plasma by liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on a reversed-phase C18 column using an isocratic mobile phase consisting of 0.1% citric acid+CH(3)CN (86:14, v/v) running at flow rate of 1.5 mL/min. The effluent was monitored at a wavelength of 280 nm. EGCG, ECG and IS were well separated from each other and free from interference from blank plasma and other components in TP as well as metabolites post-dosing. The calibration curve was constructed by plotting peak area ratio of analytes to IS vs. concentration. The method showed good linearity over range of 0.5-300 microg/mL for EGCG and 0.1-60 microg/mL for ECG (r>0.999). The intra- and inter-day precision (R.S.D.) was better than 6 and 12%, respectively. Assay accuracy was better than 94.78% for both compounds. Extraction recovery at QC samples was between 85.73 and 91.93% for EGCG and 79.08 and 86.51% for ECG. The developed method was successfully used to simultaneously measure plasma concentrations of EGCG and ECG after intravenous administration of TP to rats and yielded two typical biexponential decay concentration-time curves.     

Simultaneous determination of two Amadori compounds in Korean red ginseng (Panax ginseng) extracts and rat plasma by high-performance anion-exchange chromatography with pulsed amperometric detection

A new simple, rapid and sensitive high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAEC-PAD) was developed and validated for the simultaneous determination of two Amadori compounds, arginyl-fructose and arginyl-fructosyl-glucose in Korean red ginseng (Panax ginseng) extracts, rat plasma. Separation of the two target analytes was efficiently undertaken on CarboPac PA1 anion-exchange column with isocratic elution (400 mM sodium hydroxide and deionized water (90:10, v/v)) at flow rate 0.7 mL/min within 15 min of single chromatographic run. Under optimized conditions, the detection limits (signal-to-noise ratio equal to 3) were 20 and 25 ng/mL for arginyl-fructose and arginyl-fructosyl-glucose, respectively. Calibration curves of peak area for the two analytes were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 94.15-102.62%. This method was successfully applied to the quantification of arginyl-fructose and arginyl-fructosyl-glucose in herbal extracts and in the plasma samples from rat.     

Simultaneous determination of amoxicillin and clavulanic acid in human plasma by HPLC-ESI mass spectrometry

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-mass spectrometric (MS) method has been developed for simultaneous determination of amoxicillin and clavulanic acid in human plasma using terbutaline as internal standard. After precipitation of the plasma proteins with acetonitrile, the analytes were separated on a C(8) reversed-phase column with formic acid-water-acetonirile (2:1000:100) and detected using electrospray ionization (ESI) mass spectrometry in negative selected ion monitoring (SIM) mode. The method was validated and successfully applied to analysis of amoxicillin and clavulanic acid in clinical studies. The limit of quantitation, 0.12 microg/ml for amoxicillin and 0.062 microg/ml for clavulanic acid, was five times lower than that of the published HPLC-UV method.     

Sensitive microanalysis of gabapentin by high-performance liquid chromatography in human serum using pre-column derivatization with 4-chloro-7-nitrobenzofurazan: Application to a bioequivalence study

Most of the published methods for analysis of gabapentin, an antiepileptic agent, in human serum require automated o-phthalaldehyde derivatization of the drug and immediate injection of the unstable derivatives formed. A new, very sensitive and simple high-performance liquid chromatographic method for quantitation of the drug in human serum using 4-chloro-7-nitrobenzofurazan (NBD-Cl) as a fluorescent labeling agent is presented. In this method the sensitivity was significantly improved and the limit of quantification of 0.002 microg/ml was obtained using 100 microl serum sample and 10 microl injection. However, the LOQ can be improved by increasing the sampling volume. The procedure involved protein precipitation of serum by acetonitrile followed by derivatization with NBD-Cl. Amlodipine was used as internal standard and chromatographic separation was performed on a Shimpack CLC-C18 (150 mm x 4.6 mm) column. The fluorescence derivative of the drug was monitored at excitation and emission wavelengths of 470 and 537 nm, respectively. A mobile phase consisting of methanol and sodium phosphate buffer (0.05 M; pH 2.5) containing 1 ml/l triethylamine (65:35, v/v) was used. The calibration curve was linear over the concentration range of 0.002-15 microg/ml. No interferences were found from commonly co-administrated antiepileptic drugs. The method was applied in a randomized cross-over bioequivalence study of two different gabapentin preparations in 24 healthy volunteers.     

Simultaneous HPLC analysis, with isocratic elution, of glycyrrhizin and glycyrrhetic acid in liquorice roots and confectionery products

Glycyrrhizin (1), the main active principle of Glycyrrhiza glabra (liquorice) roots, is extensively used in herbal medicines, in pharmaceutical preparations and confectionery products. A feasible and reliable method which allows the simultaneous analysis of 1 and its aglycone, 18beta-glycyrrhetic acid (2), by means of an isocratic HPLC procedure is described. The system uses a C8 column as the stationary phase, and a mixture of acetonitrile, methanol, water and glacial acetic acid as the mobile phase. Good linearity was found in the concentration ranges 1-50 and 0.05-2.50 microg/mL for 1 and 2, respectively. A simple and rapid sample pre-treatment, based on the extraction of the two analytes with a mixture of water and ethanol, was developed for the examination of liquorice confectionery products and root samples. The HPLC method was shown to be appropriate, in terms of precision and feasibility, for the quality control of the analytes in these matrices.