Analysis of zearalenone and α-zearalenol in animal feed using high-performance liquid chromatography

Different extraction and clean-up techniques used before HPLC analysis were compared in order to obtain a reliable method for the quantitative determination of zearalenone (ZEA) and α-zearalenol (α-ZOL) in animal feed. Immunoaffinity clean-up was compared to C₁₈ and Florisil column clean-up. Extracted samples were analysed by reversed-phase HPLC with fluorescence detection (λ_ex=274 nm, λ_em=440 nm). A mobile phase of acetonitrile:water (50:50 (v/v)) and a flow-rate of 1.0 ml min⁻¹ resulted in a good separation between ZEA and α-ZOL. Using immunoaffinity clean-up the linear range was between 25 and 600 μg kg⁻¹ for ZEA and α-ZOL in maize. Intra-laboratory coefficients of variation (CV) (under repeatability conditions) were 9.16% for ZEA and 2.18% for α-ZOL. Recoveries for spiked ZEA and α-ZOL samples ranged from 89 to 110% with CVs between 5.2 and 11.2% (under within-laboratory reproducibility conditions). Using C₁₈ and Florisil solid-phase clean-up, matrix interference was too high. Therefore, naturally contaminated animal feed samples were analysed using the developed HPLC method coupled to the immunoaffinity clean-up.     

Separation and determination of terbinafine and its four impurities of similar structure using simple RP-HPLC method

A novel reversed-phase HPLC method for the simultaneous determination of active component terbinafine, its one impurity 1-methylaminomethylnaphtalene and three degradation products, β-terbinafine, Z-terbinafine and 4-methyl-terbinafine occurring in pharmaceutical formulations after long-term stability tests, was developed and validated using propylparaben as an internal standard.The chromatographic separation was performed on a NUCLEOSIL 100-5-CN column, mobile phase for separation of all compounds consisted of a mixture of tetrahydrofurane, acetonitrile and citrate buffer pH 4.50 (10:20:70, v/v/v). The analysis time was less than 32 min at flow-rate of 0.8 ml min⁻¹. UV detection was performed at 226 nm. The method was validated and system suitability parameters were investigated. Method robustness and short-term standard solution stability were verified. Limits of detection for terbinafine degradation products/impurity were from 0.023 to 0.098 μg ml⁻¹, limits of quantitation were from 0.078 to 0.327 μg ml⁻¹. The method was applicable for routine determination of terbinafine and all its found impurities of similar structure with sufficient selectivity, precision and accuracy.     

Liquid chromatography on an amide stationary phase with post-column derivatization and fluorimetric detection for the determination of streptomycin and dihydrostreptomycin in foods

The separation of streptomycin and its derivative dihydrostreptomycin using ion-pair liquid chromatography is proposed. The method is based on the use of a new stationary phase based on a ligand with amide groups and the endcapping of trimethylsilyl which avoids the appearance of tailed peaks. The isocratic mobile phase consisted of a 6:94 (v/v) acetonitrile/10 mM pentanesulfonic acid (pH 3.3) mixture at a flow-rate of 1 mL min⁻¹ and fluorescence detection involved a post-column derivatization reaction using β-naphthoquinone-4-sulfonate. Linearity, precision, recovery and sensitivity were satisfactory. The procedure was applied to the analysis of the aminoglycoside antibiotics in different types of foods, as honey, milk, egg and liver. Extraction was carried out by acidic hydrolysis to release protein-bound antibiotics. Detection limits in the food samples are 7.5 and 15 μg kg⁻¹ for streptomycin and dihydrostreptomycin, respectively.     

A simple method for the study of salbutamol pharmacokinetics by ion chromatography with direct conductivity detection

A simple method for the study of the pharmacokinetics of salbutamol using ion chromatography with direct conductivity detection without chemical suppression is presented in this paper. Baseline separation of salbutamol from plasma components was achieved by using diluted HNO₃ (2 mmol l⁻¹) as mobile phase with acetonitrile (6%, v/v) as modifier. Atenolol was utilized as internal standard. Under the optimal conditions, the linear regression coefficients of the calibration curves are 0.996 within the concentration range of 1000-3 ng ml⁻¹ salbutamol. The detection limit (signal-to-noise ratio = 3) was 1 ng ml⁻¹ salbutamol. The noncompartmental pharmacokinetic parameters for salbutamol following administration of 8 mg salbutamol to 18 subjects were tested. The results coincided most satisfactorily with the results obtained by using reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection.     

A liquid chromatography method using a monolithic column for the determination of corticoids in animal feed and animal feeding water

An HPLC-DAD method for determining corticoids in calf feed and in animal feeding water samples using a monolithic column has been developed and validated. The method optimization included the study of binary mobile phases of water and acetonitrile. The optimum separation was achieved at 40 °C, with acetonitrile:H₂O 29:71 v/v used as mobile phase and a 3 ml/min flow-rate, which resulted in their separation in about 5 min. Two reported sample procedures were applied to feed and for animal feeding water samples prior to HPLC. Method validation was carried out according to the EU criteria established for quantitative screening methods. The results indicate that this method is highly specific, reproducible and accurate. The proposed method was found to be robust and unaffected by small variations in the extraction procedure and in HPLC conditions. The developed method for the determination of corticoids in feed and water samples was also found to be suitable for different kinds of feeds and waters.     

Optimization of the simultaneous determination of imatinib and its major metabolite, CGP74588, in human plasma by a rapid HPLC method using D-optimal experimental design

A simple, rapid and specific HPLC method has been developed and validated for the simultaneous determination of imatinib, a tyrosine kinase inhibitor, and its major metabolite, CGP74588, in human plasma. The optimization of the HPLC procedure involved several variables, of which the influences of each was studied. After a series of preliminary-screening experiments, the composition of the mobile phase and the pH of the added buffer solution were set as the investigated variables, while the resolution between imatinib and CGP74588 peaks, the retention time and the imatinib peak width were chosen as the dependent variables. Applying D-optimal design, the optimal chromatographic conditions for the separation were defined. The method proved to show good agreement between the experimental data and predictive values throughout the studied parameter range.The optimum assay conditions were achieved with a Chromolith™ Performance RP-8e 100 mm × 4.6 mm column and a mixture of methanol/acetonitrile/triethylamine/diammonium hydrogen phosphate (pH 6.25, 0.048 mol L⁻¹) (20:20:0.1:59.9, v/v/v/v) as the mobile phase at a flow rate of 2 mL min⁻¹ and detection wavelength of 261 nm. The run time was less than 5 min, which is much shorter than the previously optimized methods. The optimized method was validated according to FDA guidelines to confirm specificity, linearity, accuracy and precision.     

Development and validation of HPLC method for determination of clotrimazole and its two degradation products in spray formulation

A novel simple isocratic HPLC method with UV detection for the determination of three compounds in spray solution (active component clotrimazole and two degradation products imidazole and (2-chlorophenyl)diphenylmethanol) using ibuprofen as an internal standard was developed and validated. The complications with different acido-basic properties of the analysed compounds in HPLC separation - while clotrimazole has pK_a 4.7, imidazole has pK_a 6.9 compared to relatively more acidic (2-chlorophenyl)diphenylmethanol - were finally overcome using a 3.5 μm Zorbax^® SB-Phenyl column (75 mm × 4.6 mm i.d., Agilent Technologies).The optimal mobile phase for separation of clotrimazole, degradation products imidazole and (2-chlorophenyl)diphenylmethanol and ibuprofen as internal standard consists of a mixture of acetonitrile and water (65:35, v/v) with pH* conditioned by phosphoric acid to 3.5. At a flow rate of 0.5 ml min⁻¹ and detection at 210 nm, the total time of analysis was less than 6 min.The method was applied for routine analysis (batch analysis and stability tests) in commercial spray solution.     

Determination of clarithromycin in rat plasma by HPLC-UV method with pre-column derivatization

A novel high-performance liquid chromatographic (HPLC) method using pre-column derivatization and UV detection at 275 nm for the determination of clarithromycin in rat plasma has been validated. Clarithromycin was extracted from plasma sample spiked with internal standard (erythromycin) under alkaline condition with ethyl ether and derivatizated with trimethylbromosilane. The analyses were run on a C₁₈ column, maintained at 40 °C during elution, using a mobile phase comprised of potassium dihydrogen phosphate (50 mM, pH 6.8, contained 0.7% triethylamine), acetonitrile, and methanol (30:45:25, v/v/v). The standard calibration curve for clarithromycin was linear (r² = 0.9998) over the concentration range of 0.1-10 μg ml⁻¹ in rat plasma. The limit of detection (LOD) and limit of quantitation (LOQ) was 30 ng ml⁻¹ and 0.1 μg ml⁻¹ respectively. The intra- and inter-day assay variability range was 2.6-7.4% and 3.3-8.5%, respectively. This method has been successfully applied to a pharmacokinetic study of clarithromycin in rats.     

A highly sensitive HPLC method with automated on-line sample pre-treatment and fluorescence detection for determination of reboxetine in human plasma

A fully automated, rapid and highly sensitive HPLC method with automated sample pre-treatment by column-switching system and fluorescence detection has been developed for the trace quantitative determination of the new antidepressant reboxetine (RBX) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. Paroxetine (PXT) was used as an internal standard. Plasma samples containing both RBX and PXT, after filtration, were derivatized by heating with NBD-F in borate buffer of pH 8 at 70 °C for 30 min. The derivatized plasma samples were injected into the HPLC system where an on-line sample clean up was achieved on the pre-treatment column (Co-sense Shim-pack MAYI-ODS) with a washing mobile phase (acetonitrile:2% acetic acid; 40:60, v/v) at a flow rate of 5 mL min⁻¹ for 1 min. After an automated on-line column switching to the analytical Hypersil phenyl 120A column (250 mm × 4.6 mm, 5 μm), the separation of the derivatized RBX and PXT was performed using a mobile phase consisting of sodium acetate buffer (pH 3.5):tetrahydrofuran:acetonitrile (55:35:10, v/v/v) at a flow rate of 2.0 mL min⁻¹. The eluted derivatives were monitored by a fluorescence detector set at an excitation wavelength of 470 nm and an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9995, n = 5) was found between the peak area ratio of RBX to PXT and RBX concentration in the range of 2-500 ng mL⁻¹, with limits of detection and quantification of 0.5 and 1.7 ng mL⁻¹, respectively. The intra- and inter-day precisions were satisfactory; the relative standard deviations were 2.25 and 3.01% for the intra- and inter-day precisions, respectively. The accuracy of the method proved as the mean recovery values were 100.11 ± 2.24% and 100.99 ± 2.98% for the intra- and inter-day assay runs, respectively. The proposed method involved simple and minimum sample preparation procedure and short run-time (<12 min) and therefore it can be applied to the routine therapeutic monitoring and pharmacokinetic studies of RBX.     

Development and validation of a rapid HPLC method for the determination of five banned fat-soluble colorants in spices using a narrow-bore monolithic column

This work reports a fast and simple liquid chromatographic method for the simultaneous determination of five banned fat-soluble synthetic colorants, namely Sudan I-IV and Para-Red, in spice samples. The analytes were successfully separated isocratically in less than 5 min on the new narrow bore monolithic column, FastGradient^® Chromolith (50 mm × 2.0 mm i.d.) using a mobile phase of 0.1% (v/v) HCOOH/acetonitrile (35/65%, v/v) at a flow rate of 1.5 mL min⁻¹. All colorants were detected at 506 nm. The main parameters (mobile phase composition, flow rate, injection volume) affecting the separation were studied. The proposed method was thoroughly validated in terms of linearity, LODs, precision and accuracy. The method was applied to the determination of the studied azo-dyes in various spices (paprika, chilli and mixed spice powders) after ultrasound-assisted extraction. Satisfactory recoveries, ranging from 92% to 109% were obtained.     

Ion-pair high-performance liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry for the determination of nucleotides in baby foods

A liquid chromatography with diode array detection coupled to dual electrospray atmospheric pressure chemical ionization time-of-flight mass spectrometry (HPLC/ESI-APCI-TOF-MS) method is described for the rapid determination of five monophosphate nucleotides (cytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine 5′-monophosphate, inosine 5′-monophosphate and guanosine 5′-monophosphate) in baby foods. The method is based on the deproteinisation of foods and direct analysis of nucleotides by ion-pair HPLC using isocratic elution with a mobile phase of 5% (v/v) methanol and 95% (v/v) 0.1 M formate buffer (pH 5.5) containing 0.01 M N,N-dimethylhexylamine (DMHA) at a flow-rate of 0.7 mL min⁻¹. The HPLC was hyphenated with two different detection systems, photodiode-array (DAD) and ESI-APCI-TOF-MS in negative mode. The method was validated for linearity, detection and quantitation limits, selectivity, accuracy and precision. The recoveries obtained for spiked samples were satisfactory for all the analytes. The method was successfully applied to the analysis of nucleotides in different baby and/or functional food samples, as cereals, purees and dairy products. A study was also carried out on the stability of nucleotides in acidified dairy infant food with pasteurized yoghourt and follow-on formulae samples stored at room temperature and at 30 °C.     

Simple automated generation of gradient elution conditions in sequential injection chromatography using monolithic column

The paper deals with the concept of simple automated creation of gradient profile of the mobile phase for gradient-elution sequential injection chromatography (GE-SIC). The feasibility and merits of this concept are demonstrated on the separation and simultaneous assay of indomethacin as active principle and of its two degradation products (5-methoxy-2-methylindoleacetic acid and 4-chloro-benzoic acid) in a topical pharmaceutical formulation.The GE-SIC separation was performed with a FIAlab^® 3000 SIC set-up (USA) equipped with an Onyx™ Monolithic C18 (25 mm × 4.6 mm, Phenomenex^®) column, a six-port selection valve, a 5-mL syringe pump and a fiber-optics UV CCD detector. Ketoprofen was used as an internal standard (IS). The gradient elution was achieved by automated reproducible mixing of acetonitrile and aqueous 0.2% phosphoric acid in the holding coil of the SIC system. Different profiles of the gradient elution were tested. The optimal gradient using two mobile phases 30:70 and 50:50 of acetonitrile/0.2% phosphoric acid (v/v) was achieved under the optimum flow rate 1.2 mL min⁻¹. The chromatographic resolution R between the peaks of all solutes (including the IS) was >2.00. The repeatability of retention times was characterized by the RSD values 0.18-0.30% (n = 6). Net separation time was 3.5 min and the mobile phase consumption was 4.5 mL for a single GE-SIC assay. The figures of merit of the novel GE-SIC method compared well with those of conventional HPLC.     

Dual separation mode for simultaneous determination of antihypertensive drug combinations by high-performance liquid chromatography

A simple, reproducible and efficient dual separation mode high performance liquid chromatographic (HPLC) method was developed for simultaneous determination of antihypertensive drug combinations including; hydrochlorothiazide (HCTZ), valsartan (VAL), amiloride (AML) and captopril (CAP). The newly developed Platinum™ column, which provides a dual-mode separation with its polar and non-polar sites, was used for rapid separation of these co-administered drugs. Good resolution was obtained when Platinum™ column was used compared with C₁₈ column. Additionally, simple isocratic mode with mobile phase containing methanol and 0.02 mole L⁻¹ phosphate buffer adjusted to pH 3.0 (45:55, v/v) was used for separation. The flow rate was 0.5 mL min⁻¹ and effluent was monitored at 270 nm. All the investigated drugs were completely separated within less than 6 min. The linearity range obtained for the developed HPLC method was 0.5-100 μg mL⁻¹ with detection limits of 0.13-1.2 μg mL⁻¹ for all the studied drugs. The method was validated in accordance with the requirements of ICH guidelines and shown to be suitable for intended applications. The method was successfully used for determination of the studied drugs in pure form and pharmaceutical dosage forms without prior need for separation. The method is valuable for quality control laboratories for simultaneous determination of these co-administered antihypertensive drugs in binary, ternary and quaternary mixtures.     

A three-factor Doehlert matrix design in optimising the determination of octadecyltrimethylammonium bromide by cation-exchange chromatography with suppressed conductivity detection

A simple and effective chromatographic method with suppressed conductivity detection was developed and validated to determine dissolved samples of octadecyltrimethylammonium bromide (C₁₈H₃₇N⁺Me₃Br⁻, ODTAB) for purity testing. A response surface methodology generated with a Doehlert matrix design was applied to optimize the chromatographic and detection conditions in ion-exchange chromatography (IEC) with conductivity detection in the chemical suppression mode. A three-factor Doehlert design was performed to fit a second-order model and jointly optimize the peak intensity and shorten analysis time through a global desirability function. Regenerant flow rate, volume fraction of acetonitrile in the acidic eluent and its flow rate were studied at seven, five and three levels, respectively. The optimized separation and detection conditions were accomplished by using a cation-exchange column eluted at 0.5 mL min⁻¹ with an isocratic mobile phase composed of CH₃CN and 25 mN H₂SO₄, 82/18 (v/v). Chemical suppression of ionic conductivity was performed by 100 mN tetrabutylammonium hydroxide (TBAOH) as a regenerant at a flow-rate of 4.0 mL min⁻¹. Remarkably good agreement was found between predicted and experimental values of signal intensity and chromatographic retention. With the developed method, a linear calibration curve of ODTA⁺ as bromide salt from 5 to 1000 ppm was obtained using hexadecyltrimethylammonium bromide as internal standard. The estimated limit of detection was 0.3 ppm (S/N = 3). The effectiveness of electrochemically suppressed conductivity detection of ODTA⁺ was also demonstrated, thus making easier the whole detection operation and instrumental needs as well.     

Development of an HPLC method for the determination of mexiletine in human plasma and urine by solid-phase extraction

A sensitive and specific high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of mexiletine (MEX) in human plasma and urine. It uses solid-phase extraction (SPE) followed by an automated reversed-phase HPLC with a pre-column derivatization with 4-chloro-7-nitrobenzofurazan (NBD-CI) and UV-vis Absorbance detection. The process was set as: the UV-vis Absorbance wavelength was set at 458 nm. Chromatographic separation was performed on a Phenomenex-C₁₈ Column (Aqua, 150 mm × 4.6 mm i.d. with 5 μm particle size) with the mobile phase consisting of acetonitrile and water (80:20, v/v), and the flow rate was set at 1.0 mL min⁻¹. Calibration of the overall analytical procedure gave a linear signal (r > 0.9998) over a MEX concentration range of 0.2-2.0 μg mL⁻¹ in human plasma and urine. The detection limit in plasma and urine was 0.1 μg mL⁻¹. Intra- and inter-day precision of the assay at three concentrations within this range were 0.31-2.50%. The high specificity and sensitivity have been achieved by this fast method (total run-time <6 min). The method has been successfully validated in human plasma and urine and it has been shown to be precise, accurate and reliable.     

Validated liquid chromatographic-fluorescence method for the quantitation of gemifloxacin in human plasma

A highly selective, sensitive and rapid high performance liquid chromatographic method has been developed and validated to quantify gemifloxacin in human plasma. The gemifloxacin and internal standard (ciprofloxacin) were extracted by ultrafiltration technique followed by injection into chromatographic system. Chromatographic separation was achieved on a reversed phase C₁₈ column with a mobile phase of acetonitrile:0.1% trifluoroacetic acid (20:80, v/v) using isocratic elution (at flow rate 1 mL min⁻¹). The analytes were detected at 269 and 393 nm for excitation and emission, respectively. The assay exhibited a linear range of 25-5000 ng mL⁻¹ for gemifloxacin in human plasma. The lower limit of detection was 10 ng mL⁻¹. The method was statistically validated for linearity, accuracy, precision and selectivity following FDA guidelines. The intra- and inter-assay coefficients of variation did not exceed 7.6% deviation of the nominal concentration. The recovery of gemifloxacin from plasma was greater than 97.0%. Stability of gemifloxacin in plasma was excellent with no evidence of degradation during sample processing (auto-sampler) and at least 3 months storage in a freezer at −70 °C. This validation method is applied for clinical study of the gemifloxacin in human volunteers.     

A rapid determination of propiverine and its N-oxide metabolite in human plasma by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for determination of propiverine and propiverine N-oxide metabolite in human plasma using oxybutynin as internal standard. Instead of extracting propiverine from plasma using organic solvents, which should be separated from the aqueous phase and evaporated before injecting the sample into the chromatograph, plasma sample containing propiverine and N-oxide was directly injected after precipitating proteins with acetonitrile. Numerous compounds in the plasma did not interfere with the highly specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted propiverine, N-oxide and oxybutynin within 2 min (0.1% formic acid in water/acetonitrile, 25:75, v/v). The LC-MS/MS method and an alternative LC-MS method, using methyl-t-butyl ether extraction and selected ion monitoring, were validated over 1-250 ng ml⁻¹ of propiverine and 2 to 500 ng ml⁻¹ of N-oxide, and successfully applied in a pharmacokinetic study. The lower limit of quantitation was 1 ng ml⁻¹ for propiverine and 2 ng ml⁻¹ for N-oxide in both methods.     

Validation of HPLC stability-indicating method for Vitamin C in semisolid pharmaceutical/cosmetic preparations with glutathione and sodium metabisulfite, as antioxidants

HPLC stability-indicating method was validated for Vitamin C (ascorbic acid) in semisolid pharmaceutical/cosmetic formulations containing glutathione and sodium metabisulfite, as antioxidants. The described procedure included a reliable, precise, accurate and specific method determination employing a 250 mm × 4.6 mm C₁₈ column, 0.2% metaphosphoric acid/methanol/acetonitrile (90:8:2, v/v/v) as the mobile phase and detection at 254 nm. Nicotinic and ascorbic acids were employed as standards, both presenting purity of 99.0%. Linearity was established for the ascorbic acid concentrations ranging form 1.0 to 12 μg mL⁻¹, accuracy/recovery percentage was 95.46-101.54%, precision values were 0.38 (intra-day) and 1.22% (inter-days), and LOD and LOQ were found to be 0.05 and 0.17 μg mL⁻¹, respectively. The working mobile phase elevated the ascorbic acid retention time to ≈3.5 min at a flow rate of 1.0 mL min⁻¹ and provided resolution of the active from the nicotinic acid (internal standard), degradation product (oxalic acid) and other excipients from the pharmaceutical/cosmetic preparations.     

Rapid separation and highly sensitive detection methodology for sulfonamides in shrimp using a monolithic column coupled with BDD amperometric detection

In this report, we aimed to extend our previous efforts toward the evaluation of sulfonamides (SAs) with a boron-doped diamond (BDD) electrode. We improved this method by reducing the analysis time using a monolithic column coupled with amperometric detection to determine seven sulfonamides (sulfaguanidine, sulfadiazine, sulfamethazine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline). Because of its rapid separation, low back-pressure and high separation efficiency compared to a particle-packed column, a monolithic column (100 mm × 4.6 mm) was used for sulfonamide separation. Chromatographic separation was performed in less than 8 min. The analysis was carried out using phosphate buffer (0.1 M, pH 3): acetonitrile: methanol in a ratio of 80:15:5 (v/v/v) as the mobile phase with a flow rate of 1.5 mL min⁻¹. The optimal detection potential using hydrodynamic voltammetry was found to be 1.2 V versus Ag/AgCl. The method was applied to determine seven sulfonamides in shrimp after sample preparation by solid-phase extraction. The recoveries of the sulfonamides in spiked shrimp samples at 1.5, 5 and 10 μg g⁻¹ were in the range of 81.7 to 97.5% with a relative standard deviation (R.S.D.) between 1.0 and 4.6%. Our methodology produced results that were highly correlated with HPLC-MS data. Therefore, we propose a method that can be used for the rapid, selective and sensitive evaluation of sulfonamides in contaminated food.     

Simultaneous determination of rosiglitazone and metformin in plasma by gradient liquid chromatography with UV detection

A novel, fast and simple liquid chromatographic method was developed and validated for the simultaneous determination of rosiglitazone and metformin in human plasma. The analysis was performed on a phenyl column (250 mm × 4.6 mm i.d., 5 μm) using a gradient method starting with mobile phase composed of acetonitrile:5 mM acetate buffer pH 5.5 (75:25, v/v). The flow rate was 1 mL min⁻¹. UV detection was performed at 245 nm and verapamil was used as internal standard. The total run time was less than 10 min. Sample preparation included a simple protein precipitation step with acetonitrile. Validation experiments were performed to demonstrate stability, specificity, sensitivity, linearity, accuracy, precision and robustness. The limit of quantification was 100 ng mL⁻¹ for rosiglitazone and 250 ng mL⁻¹ for metformin. The extraction recoveries were 100.02-105.0% for rosiglitazone and 105.64-103.88% for metformin. The method was applied with success to plasma samples obtained from diabetic patients undergoing treatment with rosiglitazone and metformin.