Study of the enantioseparation of imazaquin and enantioselective degradation in field soils by CZE

A fast, simple, and sensitive CE method was developed to study the separation and degradation of imazaquin enantiomers in field soils. The parameters pH and concentration of the buffer electrolyte, type and concentration of the chiral selectors, applied voltage, and temperature of the CE system were investigated. Sodium hydrogen phosphate (50 mM) at pH 10.1 containing 30 mM hydroxypropyl-beta-CD (HP-beta-CD) was found to be the suitable BGE. The reliable determination for imazaquin enantiomers was obtained at 20 kV applied voltage, 15 degrees C separation system, and detection at 214 nm. Under the optimal conditions, the LODs (S/N = 3) were 0.0097 and 0.0098 mg/kg; linearity ranged from 0.019 to 1.24 and 0.020 to 1.26 mg/kg for the first-eluting peak of imazaquin (imazaquin-I) and the second-eluting peak of imazaquin (imazaquin-II) in soil, respectively. The precision in terms of the percentage of RSD (%RSD) calculated from peak area was not greater than 5%. With the suitable characteristics of the present method, it was applied to the study of enantioselective degradation in field soil. According to the calculated values of the enantiomer ratio (ER) and the rate constant of degradation (k), the results suggested that the degradation rate of the two enantiomers were slightly different, and that the pH of the soil had a strong influence on the rate of degradation.     

Comparison of HPLC, capillary electrophoretic and direct spectrofluorimetric methods for the determination of temoporfin-poly(ethylene glycol) conjugates in plasma.

High-performance liquid chromatographic (HPLC), capillary electrophoretic (CE) and direct spectrofluorimetric methods for the determination of temoporfin-poly(ethylene glycol) 2000 conjugate (m-THPC-PEG 2000) in plasma are described and compared. m-THPC-PEG 2000 in plasma was quantitatively extracted (recovery 101-107%) with CH3OH-DMSO (4 + 1 v/v). The supernatant after centrifugation was used for HPLC, CE or direct spectrofluorimetric determination. The major drawback of the HPLC method was that it gave a broad and split peak even under gradient elution conditions, resulting in difficulty in detection and quantification. This is because m-THPC-PEG 2000 consists of a group of compounds with an average molecular mass of approximately 8680 Da owing to the wide molecular mass distribution of PEG 2000 used in the synthesis of the drug. m-THPC-PEG 2000 gave a single and relatively sharp peak when separated by CE with sodium tetraborate buffer (pH 9.45) in the presence of sodium dodecyl sulfate as the running buffer. However, this method lacks the necessary sensitivity for detecting the drug in plasma extract because of the limited sample volume that can be injected. Direct spectrofluorimetry is the method of choice because of its simplicity, specificity and sensitivity. Using an excitation wavelength of 423 nm and the specific emission maximum of 657 nm, the fluorescence intensity could be sensitively measured. The calibration curve constructed by plotting fluorescence intensity against concentration was linear within the range 1.32-1056 ng ml-1. The detection limit (S/N = 3) was 1.32 ng ml-1 and the limit of quantification (S/N = 10) was 2.24 ng ml-1. The precision and reproducibility were assessed by repeated analysis (n = 24) of spiked plasma samples at 350.8 and 699.3 ng ml-1. The RSD was 4.5% and 1.6%, respectively.     

Enantioselective determination of arotinolol in human plasma by HPLC using teicoplanin chiral stationary phase

A sensitive enantioselective high-performance liquid chromatography (HPLC) method was developed and validated to determine S-(+)- and R-(-)-arotinolol in human plasma. Baseline resolution was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar organic mobile phase consisting of methanol:glacial acetic acid:triethylamine, 100:0.1:0.1, (v/v/v) at a fl ow rate of 0.8 mL/min and UV detection set at 317 nm. Human plasma was spiked with stock solution of arotinolol enantiomers and labetalol as the internal standard. The assay involved the use of liquid-liquid extraction procedure with ethyl ether under alkaline condition for human plasma sample prior to HPLC analysis. Recoveries for S-(+)- and R-(-)-arotinolol enantiomers were in the range 93-103% at 200-1400 ng/mL level. Intra-day and inter-day precision calculated as %RSD was in the ranges 1.3-3.4 and 1.9-4.5% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges 1.2-3.5 and 1.5-6.2% for both enantiomers, respectively. Linear calibration curves in the concentration range 100-1500 ng/mL for each enantiomer showed a correlation coefficient (r) of 0.9998. The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 and 50 ng/mL (S/N = 3), respectively.     

Development of a low volume plasma sample precipitation procedure for liquid chromatography/tandem mass spectrometry assays used for drug discovery applications

The demand for high sensitivity bioanalytical methods has dramatically increased in the drug discovery stage; in addition, there has been a growing trend of reducing the sample volume that is required for these assays. A sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) procedure has been developed and tested to meet these needs. The assay requires only a low plasma sample volume (10 microL) and employs a protein precipitation procedure using a 1:6 plasma/acetonitrile ratio. The supernatant is injected directly into the LC/MS/MS system using the selected reaction monitoring (SRM) procedure for detection. A generic HPLC gradient based on a methanol/water mobile phase with a flow rate set to 0.8 mL/min was used. The test method showed very good linearity between 0.1-1000 ng/mL (R2 = 0.9737), precision (%RSD = 6-9), accuracy (%RE = -2) and reproducibility (%RSD = 11). A drug discovery IV/PO study was assayed using both the new low volume method and our standard volume (50 microL) method. The correlation of the two sets of data from the two methods was excellent (R2 = 0.9287). This new assay procedure has been successfully used in our laboratory for over 100 different rat or mouse discovery PK studies.     

Analysis of benzyldimethyldodecylammonium bromide in chemical disinfectants by liquid chromatography and capillary electrophoresis

Two novel analytical methodologies using capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) were developed and compared for the determination of benzyldimethyldodecylammonium bromide (BAB) in commercial compound chemical disinfectants. The LC analysis was performed with a Kromasil C18 (200 mm x 4.6 mm, 5 microm) column and a mobile phase of A:B = 80:20 (A: acetonitrile, B: 4 mmol/L octanesulfonic sodium--0.02 mol/L acetic sodium, adjusted with acetic acid to pH 5.2) at a flow rate of 1.0 mL/min. Detection was by ultraviolet absorption at 262 nm. The CE analysis was performed in a bare fused-silica capillary with 75 microm i.d. and total length of 46.4 cm with a buffer solution of 50% acetonitrile -50 mmol/L NaH2PO4, pH 2.24. The applied voltage was 20 kV. Detection was by ultraviolet absorption at 214 nm. Under optimized conditions, the HPLC retention time and CE migration time for BAB was 9.18 and 5.08 min, respectively. Calibration curves of peak area versus concentration gave correlation coefficients of 0.9996 for HPLC and 0.9994 for CE. The detection limits for HPLC and CE were 1.6 mg/L and 0.2 mg/L, respectively. Average recoveries at three concentration levels (50, 100, 200 mg/L for HPLC: 20, 40, 100 mg/L for CE) were 99.94 +/- 1.5, 99.64 +/- 1.3 and 99.61 +/- 0.4% for HPLC and 120.47 +/- 2.6, 102.06 +/- 8.7 and 103.05 +/- 3.0% for CE, respectively. Although both methods were shown to be suitable for the determination of BAB in commercial disinfectant compounds, CE provided analysis with less solvent purchase/disposal and better column efficiency, whereas HPLC provided superior precision.     

Fast assay of glucosamine in pharmaceuticals and nutraceuticals by capillary zone electrophoresis with contactless conductivity detection

A novel capillary electrophoresis (CE) method with contactless conductivity detection suitable for the determination of glucosamine (GlAm) and K(+) in pharmaceuticals was devised. Under the optimum conditions (aqueous 30 mM acetate buffer of pH 5.2 as the background electrolyte; voltage 30 kV; 25 degrees C), GlAm (migrating as glucosaminium cation) was well separated from K(+) that could occur in the dosage forms as excipient. The CE analysis was performed in fused-silica capillaries (50 microm i.d., 75 cm total length, 27 cm to detector) and the separation took <3 min. The calibration graphs were linear for both GlAm (100-300 microg/mL; r(2)=0.997) and K(+) (15-75 microg/mL; r(2)=0.997) when using ethanolamine (100 microg/mL) as the internal standard. The LOD values (S/N=3) were 9.3 microg/mL for GlAm and 2.9 microg/mL for K(+). The method was applied to the assay of GlAm content in various dosage forms. Intermediate precision evaluated by determining the content of GlAm in a single formulation on 3 consecutive days was characterized by RSD 2.35% (n=15). Acceptable accuracy of the CE method was confirmed by the added/found GlAm recovery experiments (recoveries 94.6-103.3%) and by statistical comparison of the results attained by the proposed CE and a reference HPLC method.     

Method of fast trace microanalysis of the chiral pesticides epoxiconazole and novaluron in soil samples using off-line flow-through extraction and on-column direct large volume injection in reversed-phase high performance liquid chromatography

An analytical method combining off-line flow-through extraction of a soil micro-sample (mass around 100 mg, packed into a short HPLC glass column) and direct on-column large-volume injection (LVI up to 1.00 mL) of a methanol-water soil extract onto a conventional C18 RP HPLC column enabled fast (within 3.5 minutes) trace micro-analysis of the relatively new chiral pesticides epoxiconazole (E) and novaluron (N), respectively. Linear calibration curves were evaluated from UV detection (230 nm) data in the range from 0.1 to 5 mg/kg in three most abundant Slovak agricultural soils. LOD (confidence band) at the levels 0.08-0.11 mg/kg and LOQ 0.4-0.6 mg/kg and LOD (S/N = 3) at the levels 0.007-0.018 mg/kg and LOQ (S/N = 10) 0.024-0.060 mg/kg, respectively, of dry soil were achieved. Recovery of pesticides in the overall LVI method including flow-through 130-200 mg soil micro-sample extraction was: for epoxiconazole from 74 to 85% and from 56% to 90% for novaluron with reproducibility within +/- 6% RSD. This fast (30 min) and simple method consists of just three steps which are short column filling with a solid micro-sample; flow-through liquid extraction and direct large-volume injection RP HPLC DAD analysis. The method is prepared for automation and further analysis of enantiomers of both investigated pesticides by achiral-chiral column switching techniques.     

Determination of levofloxacin and norfloxacin by capillary electrophoresis with electrochemiluminescence detection and applications in human urine

A novel method for the determination of norfloxacin (NOR) and levofloxacin (LVX) was developed by CE separation and electrochemiluminesence detection (ECL). The methods for capillary conditioning and the effect of solvent type were studied. Parameters affecting the CE and ECL were also investigated. Under the optimum conditions, the two analytes were well separated within 9 min. The LODs (S/N = 3) in standard solution are 4.8 x 10(-7) mol/L for NOR and 6.4 x 10(-7) mol/L for LVX, respectively. The precisions of intraday and interday are less than 4.2 and 8.1%, respectively. The LOQs (S/N = 10) in real human urine samples are 1.2 x 10(-6) mol/L for NOR and 1.4 x 10(-6) mol/L for LVX, respectively. The applicability of the proposed method was illustrated in the determination of NOR and LVX in human urine samples and the monitoring of pharmacokinetics for NOR. The recoveries of NOR and LVX at different levels in human urine samples were between 84.3 and 92.3%.     

Precision improvement for the analysis of flavonoids in selected Thai plants by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method for the analyses of kaempferol in Centella asiatica and Rosa hybrids and rutin in Chromolaena odorata was developed. The optimization was performed on analyses of flavonoids (e.g., rutin, kaempferol, quercetin, myricetin, and apigenin) and organic carboxylic acids (e.g., ethacrynic acid and xanthene-9-carboxylic acid) by investigation of the effects of types and amounts of organic modifiers, background electrolyte concentrations, temperature, and voltage. Baseline separation (R(s) = 2.83) of the compounds was achieved within 10 min in 20 mM NaH2PO4 - Na2HPO4 (pH 8.0) containing 10% v/v ACN and 6% v/v MeOH using a voltage of 25 kV, a temperature of 30 degrees C, and a detection wavelength set at 220 nm. The application of the corrected migration time (t(c)), using ethacrynic acid as the single marker, was efficient to improve the precision of flavonoid identification (% relative standard deviation (RSD) = 0.65%). The method linearity was excellent (r2 > 0.999) over 50-150 microg/mL. Precision (%RSD < 1.66%) and recoveries were good (> 96% and %RSDs < 1.70%) with detection and quantitation limits of 2.23 and 7.14 microg/mL, respectively. Kaempferol in C. asiatica and R. hybrids was 0.014 g/100 g (%RSD = 0.59%) and 0.044 g/100 g (%RSD = 1.04%), respectively, and rutin in C. odorata was 0.088 g/100 g (%RSD = 0.06%).     

Capillary electrophoresis for determination of free and albumin-bound bilirubin and the investigation of drug interaction with bilirubin-bound albumin

Capillary electrophoresis (CE) is a promising technique for assessment of free bilirubin and its interaction with albumin, as it requires only a small sample volume and provides a rapid and efficient separation of free bilirubin from its albumin-bound complex in a one-phase system. In order to maintain the equilibrium without dissociation of bilirubin from the albumin/bilirubin complex as in real clinical conditions, the coupling of CE with frontal analysis (FA) was investigated. A very large sample plug was introduced hydrodynamically into the capillary (36 cm length, 50 microm inner diameter) at 15 psi x s to develop the frontal conditions during CE separation. The working conditions for CE/FA separation of bilirubin and albumin were optimized as follows: +20 kV; running buffer, 10 mmol/L phosphate and 1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.4. The working range for bilirubin was found to vary from 5 to 206 micromol/L; precision with relative standard deviation (RSD) <2.0% for n = 3 and detection limit (signal to noise, S/N = 2) was 2 micromol/L. The residual binding capacity of a simulated cord blood serum for bilirubin was 26 mg/100 mL at pH 7.4. Bilirubin was shown to be displaced from albumin when aspirin was added. The free bilirubin concentration was found to increase to clinical significant concentrations from 11.9 to 21.1% when increasing aspirin was added in the range of 5-50 mg/100 mL, respectively. Thus, the investigation of aspirin displacement of bilirubin from albumin is clinically important and the CE/ FA method is a suitable procedure for this purpose.     

Determination of vitamin C and preservatives in beverages by conventional capillary electrophoresis and microchip electrophoresis with capacitively coupled contactless conductivity detection

The separation and detection of commonly used preservatives (benzoate, sorbate) and vitamin C by both conventional CE and microchip electrophoresis with capacitively coupled contactless conductivity detection is presented. The separation was optimized by adjusting the pH-value of the buffer and the use of hydroxypropyl-beta-CD (HP-beta-CD) and CTAB as additives. For conventional CE, optimal separation conditions were achieved in a histidine/tartrate buffer at pH 6.5, containing 0.025% HP-beta-CD and 0.1 mM CTAB. LOD ranged from 0.5 to 3 mg/L (S/N = 3) and the RSDs for migration time and peak area were less than 0.1 and 2%, respectively. A considerable reduction of analysis time can be accomplished by using microchip electrophoresis without significant loss in sensitivity under optimal separation conditions. A histidine/tartrate buffer at pH 6.5, incorporating 0.06% HP-beta-CD and 0.25 mM CTAB, gave detection limits ranging between 3 and 10 mg/L and satisfactory reproducibilities of < or =0.4% for the migration time and < or =3.5% for the peak area. The methods developed are useful for the quantitative determination of food additives in real samples such as soft drinks and vitamin C tablets.     

邻苯二甲酸为背景吸收电解质的毛细管电泳法测定低分子量肝素中的阴离子

在生产和贮存低分子量肝素的过程中,糖链上的硫酸酯基团会被水解而损失活性,因此肝素样品中常可以检测到游离的硫酸根离子,此外在生产过程中还会引入其他阴离子。为了检测低分子量肝素的质量稳定性,常用离子色谱检测低分子量肝素中游离的阴离子。但是相对分子质量较大的低分子量肝素会污染离子色谱柱和抑制器。为此发展了一种灵敏的毛细管电泳方法用于测定低分子量肝素中游离的SO~(2-)_4、Cl~-、F~-、PO~(3-)_4和OAc~-。不同于常用的背景吸收离子铬酸根,采用邻苯二甲酸根作为背景吸收电解质。与铬酸根相比,邻苯二甲酸根与所有待测阴离子电泳淌度匹配得更好,因此可以获得较窄的峰形。而且邻苯二甲酸根在230 nm检测波长下的摩尔吸光系数(4 754 L/(mol·cm))比铬酸根(254 nm,2 400 L/(mol·cm))高。因此,可以将毛细管电泳检测阴离子的灵敏度提高到与离子色谱法相当的水平。通过验证,该方法在0.002~1 mmol/L的浓度范围内具有较好的线性关系,日内(n=6)和日间(n=3)迁移时间和峰面积的相对标准偏差均小于3%。所测阴离子的检出限(S/N=3)和定量限(S/N=10)分别为0.4~0.8μmol/L和2~4μmol/L。该方法可用于监测低分子量肝素的稳定性。     

Determination of agmatine in biological samples by capillary electrophoresis with optical fiber light-emitting-diode-induced fluorescence detection

A capillary electrophoresis (CE)/optical fiber light-emitting diode (LED)-induced fluorescence detection method is developed for the determination of agmatine in biological samples. The agmatine was precolumn-derivatized with fluorescence tagging reagent, fluorescein isothiocyanate (FITC). Optimal separation and determination for agmatine were obtained with an electrophoretic buffer of 20 mM sodium borate (pH 9.2). Under the optimal conditions, the determination of agmatine was achieved in less than 4 min, and the detection limit was 4.1x10(-9) M (S/N = 3). The relative standard deviation (RSD) for 11 parallel determination of agmatine was less than 3.0%. The present CE-LED induced fluorescence detection method has been applied to detect agmatine in rat brain tissue, rat stomach tissue, human serum, and human urine. The level of agmatine in human urine was quantified by CE for the first time and found to be in the range 2.5-4.1x10(-7) M.     

Therapeutic deferoxamine and deferiprone monitoring in β‐thalassemia patients’ plasma by field‐amplified sample injection and sweeping in capillary electrophoresis

One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For β‐thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field‐amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused‐silica capillary. After liquid–liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five β‐thalassemia patients.     

Application of extraction disks in dissolution tests of clenbuterol and levothyroxine tablets by capillary electrophoresis

Sample preparation procedures using octadecyl (C₁₈) extraction disks were developed to obtain accurate and reproducible results for determinations of clenbuterol (20 μg per dose) and levothyroxine (100 μg per dose) in dissolution media of solid oral dosage forms. Preconcentration of samples allowed final concentrations of 1.1 μg/ml of clenbuterol and 4.0 μg/ml of levothyroxine to be reached prior to CE analysis. The results obtained by CE were in good agreement with those of HPLC. The precision of the migration time, peak area, peak height and accuracy were determined in both intea-day (n = 6) and inter-day (n =18) assays. Linearity was demonstrated over the ranges 0.5–80.0 μg/ml of clenbuterol and 1.0–30.0 μg/ml of levothyroxine. The mean recoveries were higher than 94.0%, ranging from 50 to 125% levels with respect to dose potencies. The proposed methodology may be generally applied to determine drugs at ng/ml concentrations.     

Comparison of the separation of nine tryptamine standards based on gas chromatography, high performance liquid chromatography and capillary electrophoresis methods

Nine tryptamines, including alpha-methyltryptamine (AMT), N,N-dimethyltryptamine (DMT), 5-methoxy-alpha-methyltryptamine (5-MeO-AMT), N,N-diethyltryptamine (DET), N,N-dipropyltryptamine (DPT), N,N-dibutyltryptamine (DBT), N,N-diisopropyltryptamine (DIPT), 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) were selected as model compounds. Comparisons of their sensitivity, selectivity, time, cost and the order of migration are described based on different separation techniques (GC, HPLC and CE, respectively). As a result, the limit of detection (S/N=3) obtained by GC/MS and LC/UV-absorption ranged from 0.5 to 15 microg/mL and 0.3 to 1.0 microg/mL, respectively. In contrast to this, based on the CZE/UV-absorption method, the limit of detection (S/N=3) was determined to 0.5-1 microg/mL. However, when the sweeping-MEKC mode was applied, it dramatically improved to 2-10 ng/mL. In the case of GC, HPLC and CE, migration times of the nine standards ranged from 11 to 15 min and 8 to 23 min by GC and HPLC, respectively; ranged from 20 to 26 min by sweeping-MEKC. The order of migration of DMT, DET, DPT and DBT follows the molecular weight, whereas the order of migration of AMT and 5-MeO-AMT (primary amines), DIPT (an isomer of DPT) and 5-methoxy-tryptamines (5-MeO-AMT, 5-MeO-DMT and 5-MeO-DIPT) can be altered by changing the separation conditions.     

Determination of L-carnitine in food supplement formulations using ion-pair chromatography with indirect conductimetric detection

A novel method for the determination of L-carnitine in food supplement formulations was developed and validated, using ion-pair chromatography with indirect conductimetric detection. The chromatographic method was based on a non-polar (C18) column and an aqueous octanesulfonate (0.64 mM) eluent, acidified with trifluoroacetic acid (5.2 mM). The retention time was 5.4 min and the asymmetry factor 0.65. A linear calibration curve from 10 to 1000 microg/ml (r= 0.99998), with a detection limit of 2.7 microg/ml (25 microl injection volume), a repeatability %RSD of 0.8 (40 microg/ml, n = 5) and reproducibility %RSD of 2.6 were achieved. The proposed method was applied for the determination of carnitine in oral solutions and capsules. No interference from excipients was found and the only pretreatment step required was the appropriate dilution with the mobile phase. Recovery from spiked samples was ranged from 97.7 to 99.7% with a precision (%RSD, n = 3) of 0.01-2.1%.     

高效液相色谱法测定鸡组织与鸡蛋中氯苯胍的残留量

建立了测定鸡组织与鸡蛋中氯苯胍残留的高效液相色谱法。试样用乙腈提取,经HLB固相萃取柱净化和流动相定容后,采用高效液相色谱法测定。以乙腈-0.05 mol/L NH4H2PO4缓冲液(6:4, v/v, pH 6.5)为流动相,流速为1.0 mL/min,于317 nm波长下检测。氯苯胍在10～1000 μg/L范围内具有良好的线性关系;方法的检出限(信噪比为3)为10 μg/L,定量限(信噪比为10)为15 μg/kg;回收率为73.1%～88.7%。该法操作简便、快速、灵敏、准确,样品处理简便易行,适用于测定鸡组织与鸡蛋中残留的氯苯胍。     

Simultaneous separation and determination of process-related substances and degradation products of venlafaxine by reversed-phase HPLC

A simple and rapid gradient RP HPLC method for simultaneous separation and determination of venlafaxine and its related substances in bulk drugs and pharmaceutical formulations has been developed. As many as four process impurities and one degradation product of venlafaxine have been separated on a Kromasil KR100-5C18 (4.6 mm x 250 mm; particle size 5 microm) column with gradient elution using 0.3% diethylamine buffer (pH 3.0) and ACN/methanol (90:10 v/v) as a mobile phase. The column was maintained at 40 degrees C and the eluents were monitored with photo diode array detection at 225 nm. The chromatographic behaviour of all the compounds was examined under variable compositions of different solvents, temperatures, buffer concentrations and pH. The method was validated in terms of accuracy, precision and linearity as per ICH guidelines. The inter- and intraday assay precision was < 4.02% (%RSD) and the recoveries were in the range of 96.19-101.14% with %RSD < 1.15%. The correlation coefficients (r2) for calibration curves of venlafaxine as well as impurities were in the range of 0.9942-0.9999. The proposed RP-LC method was successfully applied to the analysis of commercial formulations and the recoveries of venlafaxine were in the range of 99.32-100.67 with %RSD <0.58%. The method could be of use not only for rapid and routine evaluation of the quality of venlafaxine in bulk drug manufacturing units but also for the detection of its impurities in pharmaceutical formulations. Forced degradation of venlafaxine was carried out under thermal, photo, acidic, basic and peroxide conditions and the acid degradation products were characterized by ESI-MS/MS, 1H NMR and FT-IR spectral data.