Liquid chromatographic analysis of propafenone enantiomers in human plasma

A convenient and sensitive high-performance liquid chromatographic method for analysis of the enantiomers of propafenone (PPF) in human plasma was developed. Racemic propafenone and (-)-ephedrine (internal standard) were first extracted from plasma samples into a mixture of hexane-2-propanol-heptafluorobutanol (95:5:1.25, v/v). After evaporation of the organic layer, the samples were derivatized with R(-)-naphthylethyl isocyanate. The derivatization reached its maximum within 30 s at room temperature with an efficiency of 93.9 +/- 2.8% (mean +/- S.D.). The formed diastereomers were subsequently separated on a silica column with a mobile phase of hexane-2-propanol-isobutanol (96:2:2, v/v) at a flow-rate of 1.5 ml/min. The ultraviolet detection wavelength was set at 220 nm. Using 1 ml plasma, the detection limit was 6.25 ng/ml for the propafenone enantiomers. The assay was successfully employed to measure propafenone enantiomers in plasma samples of a healthy subject after oral administration of a single 150-mg dose of the racemate.     

Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers.

(+/-)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (+/-)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl-L-tartaric anhydride at 45 degrees C for 30 min. The resulting diastereomers were then resolved using a C8 analytical column with a mobile phase consisting of 0.05 M KH2PO4 (pH 3)-methanol-ethanol-triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships (r2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5-500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.     

Determination of the beta-blocker atenolol in plasma by capillary zone electrophoresis

A capillary zone electrophoretic method was optimised for the determination of the beta-blocker atenolol in plasma. Separation was performed in an uncoated silica capillary of 58.5 cm (effective length 50 cm) x 75 microm I.D., and detection was at 194 nm. The effects of the buffer (concentration and pH), the injection time, the voltage applied and the plasma clean-up procedure were studied. The determination of atenolol was achieved in less than 3 min, using an electrolyte of 50 mM H3BO3-50 mM Na2B4O7 (50:50, v/v) pH 9, injected hydrodynamically for 4 s at 50 mbar and applying a voltage of +25 kV. This method was applied to the determination of atenolol in plasma of nine hypertensive patients (male and female, aged from 39 to 73 years). Atenolol concentrations found vary from 30 to 585 ng/ml.     

Simultaneous stereoselective analysis of tramadol and its primary phase I metabolites in plasma by liquid chromatography. Application to a pharmacokinetic study in humans

This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.     

柱切换技术-高效液相色谱法快速检测大鼠血浆中的普萘洛尔对映体

建立了快速检测大鼠血浆中普萘洛尔对映体浓度的柱切换-高效液相色谱法。将自制限进填料柱作为预处理柱,通过直接进样方式,使普萘洛尔对映体在预处理柱上保留,同时除去血浆中的蛋白质等大分子;再通过柱切换技术,使普萘洛尔对映体在键合型纤维素-三(3,5-二甲基苯基氨基甲酸酯)(Chiralcel OD-RH)分析柱上得到手性拆分。通过条件优化,确定切换前预处理流动相为硼酸盐缓冲液(pH 8.5)-甲醇(95:5, v/v),流速为1.0 mL/min;切换后分析流动相为异丙醇-乙醇-0.2 mmol/L硼酸盐缓冲液(pH 8.5)(30:30:40, v/v/v),流速为0.8 mL/min;切换时间为3 min;柱温为25 ℃;检测波长为293 nm。普萘洛尔两对映体在25～500 mg/L的质量浓度范围内具有良好的线性关系(r=0.9995), 3个加标水平(50、100、250 mg/L)的平均回收率为97.89%～101.56%,日内和日间精密度均小于5%。该方法简便、快速、灵敏、准确,适于血浆样本中手性药物的药代动力学研究。     

Determination of the R-(-) and S-(+) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma by enantioselective high-performance liquid chromatography.

An enantioselective liquid chromatographic assay for the simultaneous determination of the S-(+) and R-(-) enantiomers of the monohydroxylated metabolite of oxcarbazepine in human plasma is described. The metabolite is the active principle. The method is based on the extraction of plasma with diethyl ether-dichloromethane (2:1, v/v), separation of the organic phase, evaporation of the solvent and dissolution of the residue in the mobile phase. The two enantiomers were resolved on a Chiralcel OD (250 mm x 4.6 mm I.D.) high-performance liquid chromatographic column. The separation was achieved by isocratic elution with n-hexane-2-propanol (77:23, v/v). The flow-rate of the mobile phase was 1.0 ml/min and the two enantiomers were detected by ultraviolet absorbance at 210 nm. The analytical method is suitable for the quantitative and simultaneous determination of the two enantiomers in plasma at concentrations down to 0.4 mumol/l after administration of oxcarbazepine.     

Sensitive determination of cinnarizine in human plasma by high performance liquid chromatography and fluorescence detection

Summary A sensitive high performance liquid chromatographic method has been developed for the determination of cinnarizine in human plasma. Cinnarizine and clocinizine (internal standard) were extracted from acidified plasma (pH 4.7) into carbon tetrachloride and the organic layer was evaporated. The products were separated on a Microspher C18 (3 m) column, using a mixture of 0.04 % triethylamine in 0.01 M ammonium dihydrogen phosphate (NH₄H₂PO₄), pH adjusted to 4.2 with orthophosphoric acid (H₃PO₄), and acetonitrile (2080, v/v) as mobile phase, at a flow rate of 1 ml/min at 40°C. Fluorescence detection (_ex = 245 nm, _em = 310 nm) was used; the detection limit was 0.5 ng/ml under the conditions used, and the calibration curve linear in the concentration range evaluated (1–60 ng/ml). The assay has been used to measure cinnarizine concentrations in plasma after oral administration to volunteers.     

Simultaneous determination of ketoprofen enantiomers and probenecid in plasma and urine by high-performance liquid chromatography.

A rapid, sensitive, stereospecific reversed-phase high-performance liquid chromatographic method was developed for simultaneous quantitation of ketoprofen enantiomers, probenecid and their conjugates in biological fluids. Following addition of the internal standard, indoprofen, the constituents were extracted into isooctane-isopropanol (95:5), water-washed, extracted with chloroform, then evaporated and the residue sequentially derivatized with ethyl chloroformate and L-leucinamide hydrochloride. The formed diastereomers were chromatographed on a reversed-phase column with a mobile phase of 0.06 M KH2PO4-acetonitrile-triethylamine (65:35:0.1) at a flow-rate of 1 ml/min and a detection wavelength of 275 nm. The minimum quantifiable concentration was 0.5 micrograms/ml in 100 microliters of rat plasma and urine samples. The intra- and inter-day coefficients of variation for this method are less than 10%. The assay is successfully applied to a pharmacokinetic study. The simultaneous analysis of probenecid with several other non-steroidal anti-inflammatory drugs was also successful.     

Achiral and chiral high-performance liquid chromatographic determination of flubendazole and its metabolites in biomatrices using UV photodiode-array and mass spectrometric detection

Flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, belongs to the group of benzimidazole anthelmintics, which are widely used in veterinary and human medicine. The phase I flubendazole biotransformation includes a hydrolysis of the carbamoyl methyl moiety accompanied by a decarboxylation (hydrolysed flubendazole) and a carbonyl reduction of flubendazole (reduced flubendazole). Flubendazole is a prochiral drug, hence a racemic mixture is formed during non-stereoselective reductions at the carbonyl group. Two bioanalytical HPLC methods were developed and validated for the determination of flubendazole and its metabolites in pig and pheasant hepatic microsomal and cytosolic fractions. Analytes were extracted from biomatrices into tert-butylmethyl ether. The first, achiral method employed a 250 mm x 4 mm column with octylsilyl silica gel (5 microm) and an isocratic mobile phase acetonitrile-0.025 M KH(2)PO(4) buffer pH 3 (28:72, v/v). Albendazole was used as an internal standard. The whole analysis lasted 27 min at a flow rate of 1 ml/min. The second, chiral HPLC method, was performed on a Chiralcel OD-R 250 mm x 4.6 mm column with a mobile phase acetonitrile-1 M NaClO(4) (4:6, v/v). This method enabled the separation of both reduced flubendazole enantiomers. The enantiomer excess was evaluated. The column effluent was monitored using a photodiode-array detector (scan or single wavelength at lambda=246 nm). Each of the analytes under study had characteristic UV spectrum, in addition, their chemical structures were confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) experiments. Stereospecificity in the enzymatic carbonyl reduction of flubendazole was observed. While synthetic racemic mixture of reduced flubendazole was separated to equimolar amounts of both enantiomers, practically only one enantiomer was detected in the extracts from all incubates.     

Determination of tertatolol enantiomers in biological fluids by high-performance liquid chromatography.

A stereospecific high-performance liquid chromatographic method for the quantification of (-)- and (+)-tertatolol in plasma and urine is described. The method involves solid-phase extraction followed by derivatization with S(+)-naphthylethylisocyanate to form the urea derivative, which is more sensitive to fluorescence detection. The separation of the diastereomeric derivatives was performed by reversed-phase high-performance liquid chromatography. Fluorimetric detection (lambda excitation = 220 nm, lambda emission = 320 nm) allows the quantification of tertatolol enantiomers down to 6 ng/ml. The assay was used to study the pharmacokinetic profile of tertatolol enantiomers following oral administration of racemic tertatolol; preliminary results suggest enantioselective absorption and/or disposition of tertatolol.     

Chiral analysis of baclofen by alpha-cyclodextrin-modified capillary electrophoresis and laser-induced fluorescence detection

An enantioselective method for baclofen (4-amino-3-p-chlorophenylbutyric acid) based on capillary electrophoresis (CE) separation and laser-induced fluorescence (LIF) detection has been developed. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of the nonfluorescent drug. alpha-Cyclodextrin (alpha-CD) was included in the buffer as a chiral selector for the separation of NDA-labeled S-(+)- and R-(-)-baclofen. Optimal resolution and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) containing 7 mM alpha-CD and a He-Cd laser (lambda ex = 442 nm, lambda em = 500 nm). Combined with a simple cleanup procedure, this method can be applied to the analysis of baclofen enantiomers in human plasma. The relative standard deviation (RSD) values on peak areas of a plasma sample containing 1.0 microM racemic baclofen were 6.4 and 4.9% (n = 8) for the S-(+)- and R-(-)-enantiomer, respectively. The RSD value on migration times of both enantiomers was 0.5% (n = 8). Calibration graphs for S-(+)- and R-(-)-baclofen in plasma showed a good linearity (r > or = 0.999) in the concentration range of 0.1-2.0 microM. The limit of detection of baclofen in plasma was about 10 ng/mL.     

High-performance liquid chromatographic determination of 1,2-diethyl-3-hydroxypyridin-4-one and its 2-(1-hydroxyethyl) metabolite in rat blood.

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the analysis of 1,2-diethyl-3-hydroxypyridin-4-one (CP94, I) and its 2-(1-hydroxyethyl) metabolite (II) in rat blood is described. I, II and the internal standard, 1-propyl-2-ethyl-3-hydroxypyridin-4-one (CP95, III) were extracted into dichloromethane (3 x 5 ml, with the addition of 1 g of sodium chloride) from blood (0.25 ml plus 0.75 ml of pH 7.0 morpholinopropanesulphonic acid buffer). Extractability approached 100% for I and III, and approximately 65% for II under these conditions. Chromatographic analysis was carried out using a Hypercarb porous graphitised carbon HPLC column (10 cm x 0.46 cm). The mobile phase was 14:86 (v/v) acetonitrile-NaH2PO4 buffer (10 mM, containing 2 mM EDTA, pH adjusted to 3 with phosphoric acid) and detection was by ultraviolet at 280 nm. Calibration curves were linear (correlation coefficient greater than 0.99) and reproducible over the concentration range 0-80 micrograms/ml and the coefficient of variation was less than 16% even at low (1 microgram/ml) concentrations. The minimum quantifiable level was 0.5 microgram/ml for both I and II.     

Rapid, sensitive and fully automated high-performance liquid chromatographic assay with fluorescence detection for sulmazole and metabolites

Sulmazole (2-[(2-methoxy-4-methylsulfinyl)phenyl]-3H-imidazo [4,5-b] pyridine; AR-L 115 BS) and two metabolites (sulfide, sulfone) were quantified from directly injected body fluids (plasma, urine, bile) after high-performance liquid chromatographic separation. No internal standard is needed, which is particularly advantageous when fluorescence detection is established. After automated pre-column enrichment on Corasil C18 (37-50 microns), the parent compound and biotransformation products could be backflushed and chromatographed on ODS-Hypersil (5 microns) with a mixture of 0.075 mol/l phosphate buffer-acetonitrile (2:1), an elution rate of 2.0 ml/min and fluorimetric detection (lambda ex = 330 nm; lambda em = 370 nm). A hydroxylated metabolite of sulmazole which occurs preferentially in urine (and bile) can be quantified in the above-mentioned solvent system diluted 1:1 with water, but with different fluorescence characteristics (lambda ex = 345 nm; lambda em = 515 nm). The assay was linear in the range 8-1000 ng/ml. The lower limit of detection was about 8 ng/ml or 80 pg with coefficients of variation between 0.4 and 5.8% for sulmazole.     

Determination of (S)-(-)-cathinone and its metabolites (R,S)-(-)-norephedrine and (R,R)-(-)-norpseudoephedrine in urine by high-performance liquid chromatography with photodiode-array detection.

A high-performance liquid chromatographic (HPLC) procedure with photodiode-array detection (DAD) is described for the determination of (S)-(-)-cathinone (S-CA) and its metabolites (R,S)-(-)-norephedrine (R-NE) and (R,R)-(-)-norpseudoephedrine (R-NPE) in urine. Extraction and clean-up of 1-ml urine samples were performed on a cyano-bonded solid-phase column using (+/-)-amphetamine as internal standard. The concentrated extracts were separated on a 3-microns ODS-1 column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was done at 192 nm. The detection limits for S-CA and R-NE/R-NPE in urine were 50 and 25 ng/ml, respectively. The differentiation of the enantiomers of cathinone and norephedrine was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate to the corresponding diastereomers followed by HPLC-DAD on a 5-microns normal-phase column. The R and S enantiomers of norpseudoephedrine were determined by gas chromatography-mass spectrometry after on-column derivatization with (S)-(-)-N-trifluoroacetylprolyl chloride. Following a single oral dose of 0.5 mg/kg of S-CA, the concentrations found in urine ranged from 0.2 to 3.8 micrograms/ml of S-CA, from 7.2 to 46.0 micrograms/ml of R-NE and from 0.5 to 2.5 micrograms/ml of R-NPE.     

Determination of zolmitriptan enantiomers in rat liver microsomes by chiral high performance liquid chromatography with fl uorescence detection

A selective chiral high performance liquid chromatographic method was developed and validated to separate and quantify the enantiomers of a new potent selective 5-HT(1B/1D) receptor partial agonist, S-zolmitriptan, and its antipode in rat liver microsomes induced with beta-naphtho flavone. S- and R-zolmitriptan were extracted from rat hepatic microsomal incubates with chloroform/isopropanol (75:25, v/v), and were separated on a narrow-bore enantioselective normal phase Chiralpak AD-H column (250 x 0.46 mm) with hexane-isopropanol-triethylamine (72/28/0.25, v/v/v) as mobile phase and fluorescence detection with emission at 350 nm and excitation at 291 nm. The calibration curves were linear for R- and S-zolmitriptan concentration over the range 0.1-5.0 microg/mL (r = 0.9996 and 0.9999), and the limits of quantitation were 0.1 microg/mL. The metabolism and interaction of the enantiomers of zolmitriptan in treated hepatic microsomes were investigated using chiral HPLC. There was significant difference between the disposition of the S- and R-zolmitriptan when racemic zolmitriptan or single enantiomers of zolmitriptan were incubated for 5, 10 and 20 min, suggesting that the metabolism of zolmitriptan in rat liver microsomes is enantioselective. In addition, there was also a significant difference between the IC(50) of R- to S-zolmitriptan and S- to R-zolmitriptan (IC(50S/R)/IC(50R/S) = 45.2). This indicated that the disposition process favored the S-form of zolmitriptan.     

Sensitive high-performance liquid chromatographic determination of famotidine in plasma. Application to pharmacokinetic study.

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of famotidine in human plasma is described. Clopamide was used as the internal standard. Plasma samples were extracted with diethyl ether to eliminate endogenous interferences. Plasma samples were then extracted at alkaline pH with ethyl acetate. Famotidine and the internal standard were readily extracted into the organic solvent. After evaporation of ethyl acetate, the residue was analysed by HPLC. The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-water (12:88, v/v) containing 20 mM disodium hydrogenphosphate and 50 mM sodium dodecyl sulphate, adjusted to pH 3. The HPLC microbore column was packed with 5 microns ODS Hypersil. Using ultraviolet detection at 267 nm, the detection limit for plasma famotidine was 5 ng/ml. The calibration curve was linear over the concentration range 5-500 ng/ml. The inter- and intra-assay coefficients of variation were found to be less than 10%. Applicability of the method was demonstrated by a bioavailability/pharmacokinetic study in normal volunteers who received 80 mg famotidine orally.     

Stereoselective determination of R(-)- and S(+)-MK-571, a leukotriene D4 antagonist, in human plasma by chiral high-performance liquid chromatography.

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(-)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D4 antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine-acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine-pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r2 greater than 0.999) for concentrations of enantiomers of 1 from 0.05 micrograms/ml, the lowest quantitation limit, up to 2.5 micrograms/ml. Intra-day coefficients of variation at 0.05 microgram/ml were 2.4% for the R(-)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(-)- and S(+)-1 were 2.6 and 3.6%, respectively. The utility of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.     

Indirect resolution of baclofen enantiomers from pharmaceutical dosage form by reversed-phase liquid chromatography after derivatization with Marfey's reagent and its structural variants

A high-performance liquid chromatographic (HPLC) method was developed for chiral assay of baclofen enantiomers in pharmaceutical formulations using an indirect approach. Baclofen enantiomers were derivatized with Marfey's reagent (FDNP-L-Ala-NH2) and its structural variants FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2. The resultant diastereomers were separated on RP-TLC [triethylammonium phosphate buffer (pH 4.0, 50 mm)-acetonitrile, 50:50] and on a C18 column using a linear gradient (45 min) of acetonitrile and 0.01% aqueous trifluoroacetic acid (TFA) with UV detection at 340 nm. The differences in the retention times (Delta t R) of diastereomers due to the five chiral reagents were compared. The maximum and minimum difference in retention times between separated diastereomers was for FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2, respectively. The effect of flow rate, acetonitrile content and TFA concentration on resolution was studied. The method was validated for linearity, repeatability, limit of detection and limit of quantification.     

HPLC method for the simultaneous determination of atenolol and chlorthalidone in human breast milk

A high-performance liquid chromatographic method was optimized and validated for the determination of atenolol and chlorthalidone (CT) in human breast milk. The milk samples were extracted and purified using ACN and phosphoric acid for precipitation of proteins followed by removal of ACN and milk fats by extraction with methylene chloride. The samples were applied, after an extraction procedure, to a cyanide column using a mobile phase consisting of ACN/water (35:65 v/v) and buffered at pH 4.0 with flow rate of 1.0 mL/min. Quantitation was achieved with UV detection at 225 nm using guaifenesin as the internal standard. The effectiveness of protein precipitation and clean up procedure were investigated. The method was validated over the range of 0.3-20 microg/mL for atenolol and 0.25-5 microg/mL for CT.