A simple high-performance liquid chromatographic method for the estimation of boswellic acids from the market formulations containing Boswellia serrata extract

A simple, rapid, and reproducible reverse-phase high-performance liquid chromatographic method is developed for the estimation of boswellic acids, the active constituents in Boswellia serrata oleo-gum resin. The chromatographic separation is performed using a mobile phase consisting of acetonitrile-water (90:10, % v/v) adjusted to pH 4 with glacial acetic acid on a Kromasil 100 C18 analytical column with flow rate of 2.0 mL/min and detection at 260 nm. The elution times are 4.30 and 7.11 min for 11-keto beta-boswellic acid (11-KBA) and 3-acetyl 11-keto beta-boswellic acid (A-11-KBA), respectively. The calibration curve is linear in the 11.66-58.30 microg/mL and 6.50-32.50 microg/mL range for 11-KBA and A-11-KBA, respectively. The limits of detection are 2.33 microg/mL and 1.30 microg/mL for 11-KBA and A-11-KBA, respectively. The mean recoveries are 98.24% to 104.17% and 94.12% to 105.92% for 11-KBA and A-11-KBA, respectively. The inter- and intra-day variation coefficients are less than 5%. The present method is successfully applied for the estimation of boswellic acids from the market formulations containing Boswellia serrata extract.     

Simultaneous determination of phenylephrine hydrochloride, guaifenesin, and chlorpheniramine maleate in cough syrup by gradient liquid chromatography

A simple and reliable high-performance liquid chromatographic method was developed for the simultaneous determination of mixture of phenylephrine hydrochloride (PHENYL), guaifenesin (GUAIF), and chlorpheniramine maleate (CHLO) either in pure form or in the presence of methylparaben and propylparaben in a commercial cough syrup dosage form. Separation was achieved on a C8 column using 0.005 M heptane sulfonic acid sodium salt (pH 3.4 +/- 0.1) and acetonitrile as a mobile phase by gradient elution at different flow rates, and detection was done spectrophotometrically at 210 nm. A linear relationship in the range of 30-180, 120-1800, and 10-60 microg/mL was obtained for PHENYL, GUAIF, and CHLO, respectively. The results were statistically analyzed and compared with those obtained by applying the British Pharmacopoeia (2002) method and showed that the proposed method is precise, accurate, and can be easily applied for the determination of the drugs under investigation in pure form and in cough syrup formulations.     

Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems

A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed and validated for the determination of insulin in nanoparticulate dosage forms. Its application for the development and characterization of insulin-loaded nanoparticulates composed of polyelectrolytes has also been carried out. A reversed-phase (RP) C18 column and gradient elution with a mobile phase composed of acetonitrile (ACN) and 0.1% aqueous trifluoroacetic acid (TFA) solution at a flow rate of 1 mL/min was used. Protein identification was made by UV detection at 214 nm. The gradient changed from 30:70 (ACN:TFA, v/v) to 40:60 (v/v) in 5 min followed by isocratic elution at 40:60 (v/v) for a further five minutes. The method was linear in the range of 1-100 microg/mL (R2 = 0.9996), specific with a good inter-day and intra-day precision based on relative standard deviation values (less than 3.80%). The recovery was between 98.86 and 100.88% and the detection and quantitation limits were 0.24 and 0.72 microg/mL, respectively. The method was further tested for the determination of the association efficiency of insulin to nanoparticulate carriers composed of alginate and chitosan, as well as its loading capacity for this protein. Encapsulant release under simulated gastrointestinal fluids was evaluated. The method can be used for development and characterization of insulin-loaded nanoparticles made from cross-linked chitosan-alginate.     

Determination and pharmacokinetic study of chlorogenic acid in rat dosed with Yin-Huang granules by RP-HPLC

A reversed-phase high-performance liquid chromatographic (RP-HPLC) method was described for the determination of chlorogenic acid (CGA) in rat plasma using protocatechuic acid as internal standard (IS). CGA in plasma was extracted with acetonitrile, which also acted as deproteinization agent. Chromatographic separation was performed on a Kromasil C18 column with methanol-0.2 m acetic acid (pH 3.0, 25:75, v/v) as mobile phase at a flow-rate of 1.0 mL/min with an operating temperature of 30 degrees C and UV detection at 300 nm. The standard curve was found to be linear over the concentration ranges of 0.4-2.5 microg/mL and 2.5-40 microg/mL, and the limit of quantification (LOQ) was 0.4 microg/mL. The analytical precision and accuracy were validated by relative standard deviation (RSD) and relative error, which were in ranges 3.14-10.78% and -2.20-5.00%, respectively. The average recovery of CGA was 87.59%. The method was successfully applied to the pharmacokinetic study of CGA in Yin-Huang granules.     

高效液相色谱法测定头孢他啶的含量及杂质

采用高效液相色谱法测定了头孢他啶的含量及杂质。以Alltima C18色谱柱(250 mm×4.6 mm,5 μm)为分离柱,以乙腈和磷酸盐缓冲溶液(pH 3.9)分别为流动相A和流动相B进行梯度洗脱,流速1.3 mL/min,柱温35 ℃,紫外检测波长255 nm。从头孢他啶药物中共分出14个杂质,且14个杂质间具有良好的分离度。头孢他啶在0.267~1069 μg/mL范围内与峰面积具有良好的线性关系(r=1.0000);其定量限(S/N=10)和最低检出限(S/N=3)分别为3.1 ng和0.93 ng。3个浓度的日内测定值的相对标准偏差(RSD）为0.72%(n=3),日间测定值的RSD为0.91%(n=3)。头孢他啶溶液在4 ℃避光条件下放置24 h保持稳定。本方法与欧洲/英国药典和日本药局方的方法比较,具有分离杂质数量多、分离度好的优点。     

Simultaneous determination of seven triterpenoids and triterpenoid saponins in Folium llicis Purpureae by high performance liquid chromatography coupled with evaporative light scattering detection

A new HPLC coupled with evaporative light scattering detection method was established for the simultaneous determination of seven triterpenoids and triterpenoid saponins in Folium Ilicis Purpureae traditional Chinese medicinal herb derived from the leaf of Ilex purpurea, namely, 3-omicron-beta-D-glucopyranosyl (1-->2)-beta-D-[6-omicron-methyl-glucuronopyranosyl]-28-omicron-beta-D-glucopyranosyl oleanolic acid (SQ-1), pedunculoside (SQ-2), 23-hydroxytormentic acid 28-omicron-beta-D-glucopyranoside (SQ-3), 23-hydroxytormentic acid (SQ-4), rutundic acid (SQ-5), ilexoside B (SQ-6), and ursolic acid (SQ-7). The optimal chromatographic conditions were achieved on a C18 column with a linear gradient elution of mobile phase A: deionized water-isopropyl alcohol-THF-acetic acid (90:10:6:1, v/v) and B: methanol-isopropyl alcohol-THF-acetic acid (90:10:6:1, v/v) at the flow rate of 1.0 mL/min; temperature for drift tube was set at 98degreesC and nitrogen flow rate was 2.8 L/min. All calibration curves of the seven compounds showed good linear regression (r2 > 0.995) within test ranges. The developed method has good repeatability for quantitation of all analytes interested with overall intraday and interday variation of less than 4.1%. The LOD (S/N = 3) and LOQ (S/N = 10) were less than 0.13 and 0.26 microg/mL, respectively, for all analytes. The established method was successfully used to evaluate the quality of Folium Ilicis Purpureae samples of different collections.     

Estimation of pioglitazone hydrochloride and metformin hydrochloride in tablets by derivative spectrophotometry and liquid chromatographic methods

Two simple and accurate methods of analysis to determine pioglitazone hydrochloride (PIO) and mefformin hydrochloride (MET) in combined dosage forms were developed using second-derivative spectrophotometry and reversed-phase liquid chromatography (LC). PIO and MET in combined preparations (tablets) were quantified using the second-derivative responses at 227.55 nm for PIO and 257.25 nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile (30 + 70). The calibration curves were linear [correlation coefficient (r) = 0.9984 for PIO and 0.9986 for MET] in the concentration range of 8-40 microg/mL for PIO and 4-12 microg/mL for MET. In the LC method, analysis was performed on a Hypersil ODS-C18 column with 5 microm particle size using the mobile phase acetonitrile-water-acetic acid (75 + 25 + 0.3), adjusted to pH 5.5 with liquor ammonia, at a flow rate of 0.5 mL/min. Measurement was made at a wavelength of 230 nm. Both the drugs were well resolved on the stationary phase, and the retention times were 8.5 min for PIO and 16.0 min for MET. The calibration curves were linear (r = 0.9933 for PIO and 0.9958 for MET) in the concentration range of 4-20 microg/mL for PIO and MET. Both methods were validated, and the results were compared statistically. They were found to be accurate, precise, and specific. The methods were successfully applied to the estimation of PIO and MET in combined tablet formulations.     

Validated stability-indicating HPLC-UV method for simultaneous determination of glipizide and four impurities

A selective stability-indicating HPLC-UV method for simultaneous determination of glipizide and four impurities (DPs I-IV) formed under hydrolytic conditions was developed and validated. The drug and impurities were resolved on an XTerra C18 column (250 x 4.5 mm id) in a single gradient run using buffer (0.005 M KH2PO4; pH 3.0)-methanol (60 + 40, v/v; mobile phase A) and (20 + 80, v/v; mobile phase B) at a flow rate of 0.5 mL/min with 230 nm detection wavelength. The method was linear across concentration ranges of 0.2-100, 0.1-100, 0.5-100, 0.2-100, and 0.1-50 microg/mL for glipizide and DPs I-IV, respectively. The RSD for intraday and interday precision for the drug and impurities was < 1 and < 1.2%, respectively. Satisfactory recoveries (96.58-99.97%) of each of the three concentrations selected across the linearity range of each analyte were obtained, proving the method was sufficiently accurate. The LOD was 0.07, 0.05, 0.16, 0.08, and 0.05 microg/mL and the LOQ was 0.20, 0.14, 0.50, 0.23, and 0.14 microg/mL for the drug and DPs I-IV, respectively. Each peak was resolved with resolution of > 2 from the nearest peak. Insignificant changes in retention time (< 4%) and calculated amount (< 1.65%) of drug and each impurity upon small but deliberate changes in various chromatographic parameters were observed, suggesting the method was robust. The method was applied successfully to stability testing of glipizide tablets.     

Validation of a high-performance liquid chromatographic method for the simultaneous determination of tramadol and its impurities in oral drops as a pharmaceutical formulation

The novel, rapid high performance liquid chromatographic method for the determination of tramadol hydrochloride and its three impurities was developed and validated. The method can simultaneously assay potassium sorbate, used as preservative, and saccharin sodium, used as sweetener in tramadol pharmaceutical formulation. The separation was carried out on a C(18) XTerra (150 mm x 4.6 mm, 5 mm) column using acetonitrile-0.015 M Na(2)HPO(4) buffer (2:8, v/v) as mobile phase (pH value 3.0 was adjusted with orthophosphoric acid) at a flow rate 1.0 ml min(-1), temperature of the column 20 degrees C and UV detection at 218 nm. The method was found to be linear (r > 0.999) in the range of 0.05-0.8 mg ml(-1) for tramadol hydrochloride, 0.1-1.2 mg ml(-1) for impurities B and C and for impurity A (r > 0.995) in the range 0.15-2.4 mg ml(-1). The low RSD values indicate good precision and high recovery values indicate excellent accuracy of the HPLC method. Developed method was successfully applied to the determination of tramadol hydrochloride, its investigated impurities and potassium sorbate in commercial formulation. The recovery of tramadol hydrochloride was 98.25% and RSD was 1.80%. The method is rapid and sensitive enough to be used to analyse Trodon oral drops.     

Gradient HPLC-diode array detector stability-indicating determination of lidocaine hydrochloride and cetylpyridinium chloride in two combined oral gel dosage forms

A simple, rapid, and selective HPLC-diode array detector method was developed for the simultaneous determination of lidocaine hydrochloride (LD) and cetylpyridinium chloride (CPC) in two combined pharmaceutical formulations. Effective chromatographic separation was achieved on a Zorbax SB-C8 (4.6 x 250 mm, 5 microm particle size) column with gradient elution using a mobile phase composed of 0.05 M phosphoric acid and acetonitrile. The gradient elution started with 25% (v/v) acetonitrile, ramped up linearly to 85% in 5 min, and then was constant until the end of the run. The mobile phase was pumped at a flow rate of 1.2 mL/min. The multiple wavelength detector was set at 214 and 258 nm, and quantification of the analytes was based on measuring their peak areas. The retention times for LD and CPC were about 3.4 and 7.3 min, respectively. The reliability and analytical performance of the proposed HPLC procedure were statistically validated with respect to linearity, range, precision, accuracy, selectivity, robustness, LOD, and LOQ. Calibration curves were linear in the range of 5-200 and 10-400 microg/mL for LD and CPC, respectively, with correlation coefficients > 0.999. The proposed method was proven to be stability-indicating by the resolution of the two analytes from the related substance and potential impurity (2,6-dimethylaniline) as well as from forced-degradation products. The validated HPLC method was extended to the analysis of LD and CPC in two combined oral gel preparations for which the two analytes were successfully resolved from the pharmaceutical adjuvants and quantified with recoveries not less than 97.9%.     

HPLC determination and pharmacokinetic studies of salvianolic acid B in rat plasma after oral administration of Radix Salviae Miltiorrhizae extract

A precise and reproducible HPLC method has been established and validated for determination of salvianolic acid B (SalB) in rat plasma after oral administration of Radix Salviae Miltiorrhizae extract. Liquid-liquid extraction was adopted for the sample preparation. Separation was accomplished on a C(18) column with a linear gradient elution consisting of acetonitrile and aqueous phosphoric acid. Ultraviolet detection was at 280 nm. The method was validated over the concentration range 10.8-259.4 microg/mL using 1 mL of plasma. The assay was linear over this concentration range with a coefficient of variation less than 7%. The extraction recovery of SalB was within the range 71-83% with RSD 11%. The mean recovery of the internal standard was 84% (n = 6) with RSD of 5.6%. This method is suitable to determine SalB in plasma and to investigate the pharmacokinetics of SalB.     

Stability-indicating high-performance column liquid chromatography and high-performance thin-layer chromatography methods for the determination of olopatadine hydrochloride in tablet dosage form

This paper describes two simple, specific, accurate, and precise methods for estimation of olopatadine hydrochloride (OLO) in tablet dosage form. The first method is a stability-indicating isocratic RP-HPLC method. The analysis is performed on an RP-18 column using 0.1% orthophosphoric acid (adjusted to pH 4.5 with triethylamine)-acetonitrile (75 + 25, v/v) mobile phase at a flow rate of 1 mL/min. Paracetamol (PAR) was selected as the internal standard. Retention times of OLO and PAR were 11.30 +/- 0.02 and 4.70 +/- 0.03 min, respectively. For the HPTLC method, precoated silica gel 60 F254 aluminum sheets were used as the stationary phase; the mobile phase was methanol-chloroform-ammonia (8 + 2 + 0.1, v/v/v). The detection of the analyte band was carried out at 301 nm, and its Rf value was 0.46 +/- 0.03. The analytical methods were validated according to International Conference on Harmonization guidelines. Linear regression analysis data for the calibration plots showed a good linear relationship between response and concentration in the range of 0.1-1 microg/mL and 0.1-0.9 microg/band for HPLC and HPTLC, respectively.     

Direct method for determination of Sudan I in FD&C Yellow No. 6 and D&C Orange No. 4 by reversed-phase liquid chromatography

A reversed-phase liquid chromatographic method was developed to determine parts-per-million and higher levels of Sudan 1, 1-(phenylazo)-2-naphthalenol, in the disulfo monoazo color additive FD&C Yellow No. 6 and in a related monosulfo monoazo color additive, D&C Orange No. 4. Sudan I, the corresponding unsulfonated monoazo dye, is a known impurity in these color additives. The color additives are dissolved in water and methanol, and the filtered solutions are directly chromatographed, without extraction or concentration, by using gradient elution at 0.25 mL/min. Calibrations from peak areas at 485 nm were linear. At a 99% confidence level, the limits of determination were 0.008 microg Sudan I/mL (0.4 ppm) in FD&C Yellow No. 6 and 0.011 microg Sudan I/mL (0.00011%) in D&C Orange No. 4. The confidence intervals were 0.202 +/- 0.002 microg Sudan I/mL (10.1 +/- 0.1 ppm) near the specification level for Sudan I in FD&C Yellow No. 6 and 20.0 +/- 0.2 microg Sudan I/mL (0.200 +/- 0.002%) near the highest concentration of Sudan I found in D&C Orange No. 4. A survey was conducted to determine Sudan I in 28 samples of FD&C Yellow No. 6 from 17 international manufacturers over 3 years, and in a pharmacology-tested sample. These samples were found to contain undetected levels (16 samples), 0.5-9.7 ppm Sudan I (0.01-0.194 microg Sudan I/mL in analyzed solutions; 11 samples including the pharmacology sample), and > or =10 ppm Sudan I (> or = 0.2 microg Sudan I/mL; 2 samples). Analyses of 21 samples of D&C Orange No. 4 from 8 international manufacturers over 4 years found Sudan I at undetected levels (8 samples), 0.0005 to < 0.005% Sudan I (0.05 to < 0.5 microg Sudan I/mL in analyzed solutions; 3 samples, including a pharmacology batch), 0.005 to <0.05% Sudan I (0.5 to <5 microg Sudan I/mL; 9 samples), and 0.18% Sudan I (18 microg Sudan I/mL; 1 sample).     

高效液相色谱法同时测定化妆品中的7种磺胺及甲硝唑和氯霉素

建立了化妆品中7种磺胺(磺胺醋酰、磺胺吡啶、磺胺甲基嘧啶、磺胺二甲嘧啶、磺胺甲氧嘧啶、磺胺间甲氧嘧啶、磺胺甲基异唑)和甲硝唑及氯霉素的高效液相色谱测定方法。样品经0.1%甲酸水溶液-乙腈(体积比为8∶2)混合液超声提取后进行液相色谱分析。方法的定量检测限为3～80 μg/g,7种磺胺在20～200 μg/mL时,甲硝唑及氯霉素在40～400 μg/mL时方法的线性关系良好(r≥0.9993)。加标回收率为83.8%～105.3%(7种磺胺的添加水平为50 μg/mL和150 μg/mL,甲硝唑及氯霉素的添加水平为100 μg/mL和300 μg/mL),其相对标准偏差均小于5%。     

微乳液相色谱法测定人血浆中丙泊酚浓度

建立了基于微乳液相色谱(MELC)的人血浆中丙泊酚浓度的测定方法。采用Hypersil BDS C18色谱柱分离,并考察了微乳流动相中各组分对溶质洗脱的影响。优化的色谱条件: 以0.5%醋酸（含有3.0%十二烷基硫酸钠（SDS）,0.8%正庚烷,6.0%正丁醇）微乳为流动相,流速为1.0 mL/min,荧光检测器激发波长(λex)为274 nm、发射波长(λem)为312 nm,柱温为室温。人血浆样品用流动相稀释并离心后,直接进样分析。丙泊酚在0.25～10 μg/mL质量浓度范围内呈良好的线性关系,方法的回收率为(98.2±1.9)%～(104.6±2.2)%;日内测定峰面积的相对标准偏差(RSD)为1.42%～2.43%,日间测定峰面积的RSD为2.75%～4.79%。该方法简便可行、重复性好,可用于人血浆中丙泊酚浓度的测定。     

Rapid high-performance liquid chromatography method for determination of pregabalin in a pharmaceutical dosage form following derivatization with fluorescamine

A simple, fast, and sensitive HPLC method was developed and validated for the evaluation of pregabalin in a pharmaceutical dosage form using fluorescamine as a derivatization agent for the first time. After a precolumn derivatization (5 min, room temperature), the reaction mixture was chromatographed on a C18 column with isocratic elution using 0.2% of triethylamine in a mixture of methanol and water (10 + 90, v/v). 3-Aminopentanoic acid was used as the internal standard. Using fluorescent detection (lamdaex 395 nm, lamdaem 476 nm), a low detection limit of 0.02 microg/mL was reached. The method was linear (r > 0.999) over the lower (0.125-25 microg/mL) and higher (1.25-250 microg/mL) concentration range. The intraday and interday precision of the QC samples was < 4.3%, and the accuracy was 94.2-102.5%. The samples were stable for 24 h at 4 degrees C. The robustness study showed that the derivatization is more robust than the chromatography method. The method was applied for the analysis of pregabalin content in 25, 75, and 300 mg capsules, and a good agreement was found with the declared amount of pregabalin (the relative error did not exceed 3.2%). Finally, the method was successfully used for dissolution studies of pregabalin capsules.     

金银花不同部位中绿原酸和木犀草苷含量的测定

利用HPLC法测定金银花不同部位中绿原酸和木犀草苷。采用ODS-C18色谱柱(250 mm×4.6 mm,5μm),以甲醇-水(0.03 mol/L磷酸)为流动相进行梯度洗脱,流速:1.0 mL/min,检测波长:350 nm,柱温:室温。绿原酸浓度在0~0.4 mg/mL内与峰面积呈良好线性关系(r2=0.999 6),回收率为101.9%~105.2%,相对标准偏差为3.18%(n=6)。木犀草苷浓度在0~0.1 mg/mL内与峰面积呈良好线性关系(r2=0.999 1),平均回收率为100.5%~104.0%,相对标准偏差为3.42%(n=6)。结果表明:金银花、芽等部位绿原酸和木犀草苷的含量较高。     

A rapid derivative spectrophotometric method for simultaneous determination of naphazoline and antazoline in eye drops

A zero-crossing first-derivative spectrophotometric method is applied for the simultaneous determination of naphazoline hydrochloride and antazoline phosphate in eye drops. The measurements were carried out at wavelengths of 225 and 252 nm for naphazoline hydrochloride and antazoline phosphate, respectively. The method was found to be linear (r2>0.999) in the range of 0.2-1 microg/ml for naphazoline hydrochloride in the presence of 5 microg/ml antazoline phosphate at 225 nm. The same linear correlation (r2>0.999) was obtained in the range of 1-10 microg/ml of antazoline phosphate in the presence of 0.5 microg/ml of naphazoline hydrochloride at 252 nm. The limit of determination was 0.2 microg/ml and 1 microg/ml for naphazoline hydrochloride and antazoline phosphate, respectively. The method was successfully used for simultaneous analysis of naphazoline hydrochloride and antazoline phosphate in eye drops without any interference from excipients and prior separation before analysis.     

Pressurized capillary electrochromatographic assay of trimethoprim impurities using 1 microm particle-based columns

One micrometre silica particles, derivatized with C18, were electrokinetically packed into a 75-microm-i.d. capillary. The resulting column was evaluated for the separation of trimethoprim (TMP) and its impurities using pressurized capillary electrochromatography (pCEC), starting from a capillary liquid chromatographic (CLC) separation. These samples require gradient elution when separated by high performance liquid chromatography (HPLC), but with the new columns isocratic elution suffices for their separation by CLC or pCEC. Only 70,000 theoretical plates/m for impurity C were achieved using CLC mode at relative low pressure (78 bar) although very small particles were utilized. When a voltage above 2 kV (50 V/cm) was applied, unknown peaks appeared, which was assumed due to an electrophoretic effect with the unknown peaks resolving as a result of the applied voltage. In order to minimize these unfavorable contributions, only a low voltage was applied, still leading to higher separation performances and shorter separation times than in CLC. The optimal analyzing conditions in pCEC included a pressure of 78 bar, an applied voltage of 1 kV, and a mobile phase consisting of 80 mM sodium perchlorate (pH 3.1)/methanol (60/40, v/v). These conditions were used to separate and quantify four major impurities in TMP within 22 min. The obtained calibration curves were linear (r>0.9980) in concentration ranges between 0.005 and 0.1 mg/mL for impurities A and C; 0.02 and 0.10 mg/mL for impurity F; and 0.01 and 0.10 mg/mL for impurity H. The detection limits (S/N=3) for impurities A, C, F, and H were 0.52, 0.84, 3.18, and 2.41 microg/mL, respectively. The calibration curves were successfully applied to analyze spiked bulk samples, with mean recoveries ranging from 92% to 110%. The developed method can therefore be considered simple, rapid, and repeatable.     

Simultaneous determination of rifampicin and sulbactam in mouse plasma by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method with ultraviolet detection has been developed and validated for the simultaneous determination of rifampicin and sulbactam in mouse plasma. Plasma samples were deproteinized with acetonitrile and separated by HPLC on a RP-18 (125 x 4 mm, 5 microm) column and gradient elution with potassium dihydrogen phosphate solution (pH 4.5; 50 mm) and acetonitrile at a flow-rate of 1.0 mL/min. Rifampicin and sulbactam were monitored at 230 nm and confirmed by means of their UV spectra using a diode-array detector. The method was linear at plasma levels from 1 to 100 microg/mL for rifampicin and from 5 to 200 microg/mL for sulbactam. The limits of quantification were 0.6 microg/mL for rifampicin and 4.2 microg/mL for sulbactam. The intra- and inter-day precisions of the method (RSD) were lower than 5% for both compounds. Average recoveries of rifampicin and sulbactam from mice plasma were 98.2 and 89.3%, respectively. The developed method was successfully applied to the determination of the pharmacokinetic profile of both compounds in mice.