Differential cytotoxic effects of denitroaristolochic acid II and aristolochic acids on renal epithelial cells

Aristolochic acids (AAs), naturally occurring nephrotoxins and rodent carcinogens, are commonly found in medicinal plants such as Radix aristolochiae. The toxicity of AAs is believed to be associated with the formation of promutagenic AA-DNA adducts, and it has also been suggested that the nitro group in AAs might be important in the process. In order to investigate the role of the nitro group in AA-mediated cytotoxicity, the effects of denitroaristolochic acid II (dN-AAII), aristolochic acid II (AAII) and aristolochic acid I (AAI) on renal tubular epithelial Madin–Darby canine kidney (MDCK) cells were examined and compared. The cytotoxicity of AAI, AAII and dN-AAII was found to be time- and concentration-dependent. As determined by MTT assay, AAI was found to be most toxic in MDCK cells upon exposure for 24, 48 and 72 h, followed by AAII, and dN-AAII showed the least cytotoxicity. The effect of AAI and AAII on the integrity of cell membrane was found to be similar and appeared to be more prominent than that of dN-AAII. Based on the results obtained from the flow cytometric analysis, significant apoptosis in MDCK cells was observed with AAI and AAII at as low as 25 μmol/L following exposure for 24 h, whereas significant apoptosis was induced by dN-AAII at a much higher concentration, 300 μmol/L, suggesting that both AAI and AAII were significantly more cytotoxic than dN-AAII. In addition, the levels of reactive oxygen species (ROS) were increased following treatment with AAI, AAII and dN-AAII at concentrations of 5, 25 and 25 μmol/L, respectively, for 4 h. The results suggest that the nitro group plays an important role in AA-mediated cytotoxicity in MDCK cells and increased intracellular ROS levels may be associated, at least in part, with the cell injury observed in MDCK cells.     

Studies on the metabolism of aristolochic acids I and II

1. After oral administration of aristolochic acid I (AAI) and aristolochic acid II (AAII) to rats, the following metabolites were isolated from the urine and their structures elucidated: aristolactam I, aristolactam Ia, aristolochic acid Ia, aristolic acid I and 3, 4-methylenedioxy-8-hydroxy-1-phenanthrenecarboxylic acid (metabolites of AAI); or aristolactam Ia, aristolactam II and 3,4-methylenedioxy-1-phenanthrenecarboxylic acid (metabolites of AAII). A further metabolite of AAII having a lactam structure has not yet been isolated in pure form.

2. The metabolic pathways have been elucidated by administration of various metabolites.

3. The principal metabolite of AAI in rats was aristolactam Ia; 46% of the dose was excreted in the urine in form of this metabolite and 37% in the faeces. The other substances were minor metabolites. Those metabolites of AAII whose structures have been elucidated were minor metabolites; the largest proportion consisted of aristolactam II, which accounted for 4.6% in the urine and 8.9% in the faeces.

4. The mouse was the only animal which had the same metabolite patterns of AAI and AAII as those found in the rat. Not all the metabolites listed above were found in urine from guinea pigs, rabbits, dogs and man.     

Studies on the metabolism of aristolochic acids I and II

1. After oral administration of aristolochic acid I (AAI) and aristolochic acid II (AAII) to rats, the following metabolites were isolated from the urine and their structures elucidated: aristolactam I, aristolactam Ia, aristolochic acid Ia, aristolic acid I and 3,4-methylenedioxy-8-hydroxy-1-phenanthrenecarboxylic acid (metabolites of AAI); or aristolactam Ia, aristolactam II and 3,4-methylenedioxy-1-phenanthrenecarboxylic acid (metabolites of AAII). A further metabolite of AAII having a lactam structure has not yet been isolated in pure form. 2. The metabolic pathways have been elucidated by administration of various metabolites. 3. The principal metabolite of AAI in rats was aristolactam Ia; 46% of the dose was excreted in the urine in form of this metabolite and 37% in the faeces. The other substances were minor metabolites. Those metabolites of AAII whose structures have been elucidated were minor metabolites; the largest proportion consisted of aristolactam II, which accounted for 4.6% in the urine and 8.9% in the faeces. 4. The mouse was the only animal which had the same metabolite patterns of AAI and AAII as those found in the rat. Not all the metabolites listed above were found in urine from guinea pigs, rabbits, dogs and man.     

Development and validation of an HPLC-DAD method for bis(12)-hupyridone and its application to a pharmacokinetic study

A rapid and simple method of high performance liquid chromatography with UV detection for the quantification of bis(12)-hupyridone in rat blood has been developed and validated. Chromatographic separation was carried out in an Agilent Extend C₁₈ 5 μm column (length, 250 mm; inner diameter, 4.6 mm) using a mixture of water–acetonitrile–trifluoroacetic acid (81:19:0.04, v/v/v) as the mobile phase at a flow rate of 1 mL/min, with detection at 229 nm. The method used for the bis(12)-hupyridone quantification showed linearity for concentration range of 0.1–7.5 μg/mL with r² = 0.9991. The limit of detection and quantification of this method were 0.05 μg/mL and 0.1 μg/mL, respectively. The intra- and inter-day variations of the analysis were less than 4.22% with standard errors less than 13.3%. The developed method was successfully applied to the pharmacokinetic study of bis(12)-hupyridone after intravenous administration of 5 mg/kg and intraperitoneal administration of 10 and 20 mg/kg in rats.     

Determination of oxaceprol in rat plasma by LC–MS/MS and its application in a pharmacokinetic study

A sensitive method for the quantification of oxaceprol in rat plasma using high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed. Sample pretreatment involved a simple protein precipitation by the addition of 60 μL of acetonitrile–methanol (1:2, v/v) to 20 μL plasma sample volume. Separation was achieved on a Dikma ODS-C18 (5 μm, 150 mm × 4.6 mm) reversed-phase column at 40 °C with acetonitrile/0.1% formic acid–4 mM ammonium acetate in water (35:65,v/v) at a flow rate of 0.6 mL/min. Detection was performed using an electrospray ionization (ESI) operating in negative ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 172 → 130 (oxaceprol) and m/z 153 → 109 (protocatechuic acid, internal standard). The calibration curve of oxaceprol in plasma showed good linearity over the concentration range of 1.25–800 ng/mL. The limit of detection and limit of quantification were 0.400 ng/mL and 1.25 ng/mL, respectively. Intra- and inter-day precisions in all samples were within 15%. There was no matrix effect. The validated method was successfully applied to a preclinical pharmacokinetic study of oxaceprol in rats. After oral administration of 20 mg/kg oxaceprol to rats, the main pharmacokinetic parameters T_max, C_max, T_1/2, V_z/F and AUC_0–t were 1.4 h, 1.2 μg/mL, 2.3 h, 19.7 L/kg and 3.4 mg h/L, respectively.     

Determination of tegafur, 5-fluorouracil,gimeracil and oxonic acid in human plasma using liquid chromatography–tandem mass spectrometry

S-1 is an oral anticancer drug, which consists of tegafur (FT), gimeracil (CDHP) and potassium oxonate (Oxo) at a molar ratio of 1:0.4:1. Among these, tegafur is a prodrug, and is rapidly metabolized to the active drug, 5-fluorouracil (5-FU), in vivo. To evaluate the pharmacokinetics of S-1 in patients, LC–MS/MS methods were developed and validated for determination of FT, 5-FU, CDHP and Oxo in human plasma. FT, 5-FU and CDHP were extracted from plasma following protein precipitation, separated on a Synergi Hydro-RP column and simultaneously quantified by LC–MS/MS. The mobile phase consisted of methanol–water–ammonia–acetic acid (27:73:0.0018:0.018, v/v/v/v). The mass spectrometer was operated in negative mode using electrospray ionization. The calibration curves were linear in the range of 12.0–3000 ng/mL for FT, and 2.00–500 ng/mL for 5-FU and CDHP. The accuracy ranged from 93.1% to 110.7% and the precision ranged from 2.4% to 14.6% for each analyte. To determine Oxo in human plasma, an LC–MS/MS method employing pre-column derivatization was developed and validated. 4-Bromomethyl-7-methoxycoumarin was chosen as the derivatization reagent and [¹³C₂,¹⁵N₃]-Oxo was used as the internal standard. The MS/MS detection was operated in positive mode using an APCI source. The calibration range was 2.00–150 ng/mL. The accuracy and precision were within 95.9–99.1% and 4.4–10.0%, respectively. The validated methods were successfully applied to characterize the pharmacokinetic profiles of FT, 5-FU, CDHP and Oxo following oral administration of 60 mg S-1 tablets to patients with solid gastrointestinal tract tumors.     

Targeted metabolite analysis of Catharanthus roseus and its biological potential

Catharanthus roseus is nowadays one of the most studied medicinal plants. In this work, further knowledge on different parts of this species (leaves, stems, seeds and petals) was achieved, namely phenolics by HPLC-DAD and organic acids and amino acids by HPLC-UV. Also, the biological potential, expressed as acetylcholinesterase inhibitory activity was accessed and, in some parts, an acetylcholinesterase inhibitory capacity higher than 85% was found (IC₅₀ at 422, 442 and 2683 μg/mL in leaves, stems and petals, respectively). C. roseus aqueous extract revealed to be a rich source of phenolics, namely caffeoylquinic acids and flavonoids derivatives (up to 4127 mg/kg in stems, 4484 mg/kg in seeds, 8688 mg/kg in leaves and 41125 mg/kg in petals), organic acids (962, 6678, 25972 and 12463 mg/kg in seeds, petals, stems and leaves, respectively), such as citric acid (over 85% in some plant parts), and amino acids (31557, 39327, 50540 and 159697 mg/kg in stems, petals, seeds and leaves, respectively), of which arginine was a major compound.     

Organic anion transporter 1 (OAT1) involved in renal cell transport of aristolochic acid I

Aristolochic acids (AAs) are a family of structurally related nitrophenanthrene carboxylic acids that are present in medicinal herbs such as Aristolochia species. The organic anion transporters (OATs) of the solute carrier (SLC22) gene family located in the renal proximal tubules play a key role in the excretion of a variety of exogenous and endogenous compounds. However, it is unclear how AAs permeate into renal epithelial cells. In this regard, we investigated the role of rat OAT1 ([rOAT1] SLC22A6) in the cellular uptake of AAI in vitro and in vivo. A concentration- and time-dependent intracellular accumulation of AAI was observed in rOAT1-transfected human embryonic kidney 293 (HEK293) cells, which was 2- to 6-fold higher than the control cells. There was a significantly increased rate of cellular apoptosis in rOAT1-transfected HEK293 cells than control cells after AAI treatment. Para-aminohippuric acid (PAH) significantly reduced the intracellular accumulation of AAI in rOAT1-transfected HEK293 cells. Administration of AAI for 35 days in rats caused significantly reduced expression of OAT1 in basolateral membrane and declined renal clearance of PAH as well as renal proximal tubule injuries. These findings indicate that AAI is taken up by OAT1, which then exert its intracellular toxic effects on renal proximal tubule cells, which in turn damage functional OAT1 and may further disturb the transport of its substrates.     

Simultaneous determination of irbesartan and hydrochlorothiazide in human plasma using HPLC coupled with tandem mass spectrometry: Application to bioequivalence studies

A sensitive, specific and selective liquid chromatography/tandem mass spectrometric method has been developed and validated for the simultaneous determination of irbesartan and hydrochlorothiazide in human plasma. Plasma samples were prepared using protein precipitation with acetonitrile, the two analytes and the internal standard losartan were separated on a reverse phase C₁₈ column (50 mm × 4 mm, 3 μm) using water with 2.5% formic acid, methanol and acetonitrile (40:45:15, v/v/v (%)) as a mobile phase (flow rate of 0.70 mL/min). Irbesartan and hydrochlorothiazide were ionized using ESI source in negative ion mode, prior to detection by multiple reaction monitoring (MRM) mode while monitoring at the following transitions: m/z 296 → 269 and m/z 296 → 205 for hydrochlorothiazide, 427 → 175 for irbesartan. Linearity was demonstrated over the concentration range 0.06–6.00 μg/mL for irbesartan and 1.00–112.00 ng/mL for hydrochlorothiazide. The developed and validated method was successfully applied to a bioequivalence study of irbesartan (300 mg) with hydrochlorothiazide (12.5 mg) tablet in healthy volunteers (N = 36).     

Rapid and sensitive assay for trantinterol,a novel β2-adrenoceptor agonist,in human plasma using liquid chromatography–tandem mass spectrometry

A rapid and sensitive assay for trantinterol, a novel β₂-adrenoceptor agonist, in human plasma has been developed. Samples containing the analyte and internal standard, clenbuterol, were analyzed by liquid chromatography–tandem mass spectrometry after liquid–liquid extraction with diethyl ether:dichloromethane (60:40, v/v). Separation was performed on a Venusil MP C₁₈ column (50 mm × 4.6 mm, 5 μm) using methanol:1% formic acid (50:50, v/v) as mobile phase and monitored by multiple reaction monitoring of the precursor-to-product ion transitions of trantinterol at m/z 311.2 → 238.1 and clenbuterol at m/z 277.2 → 203.1. The total run time was only 1.5 min and the method was linear over the concentration range 1–1000 pg/mL with a lower limit of quantitation of 1 pg/mL. Intra- and inter-day precisions (relative standard deviation) were below 7% and 12%, respectively, with accuracy (relative error) below 8%. The method was successfully applied to a pharmacokinetic study involving oral administration of a 50 μg trantinterol tablet to healthy volunteers.     

Leptin and IL-8: Two novel cytokines screened out in childhood lead exposure

Lead is a toxic heavy metal with many recognized adverse health side effects. The central nervous system is the main target of lead toxicity. Although many studies on lead toxicity were conducted, the mechanism of lead toxicity remains uncertain. One possible attribution is the immature blood–brain barrier that causes lead exposure in children. Few studies have investigated the cytokine changes caused by this exposure. Novel cytokines were detected by RayBio^® Human Cytokine Antibody Array and validated by enzyme-linked immunosorbent assay. Several children were admitted to West China Second University Hospital, after a serious lead pollution event in longchang, Sichuan, China. A total of 4 children with elevated blood lead levels (BLLs) and 4 children with low BLLs were randomly chosen in the discovery set, and 40 children with elevated BLLs and 40 children with low BLLs were included in the validation set. Leptin and interleukin-8 (IL-8) were identified to be significantly different between children with elevated and low BLLs via RayBio^® Human Cytokine Antibody Array. In the validation set, IL-8 was higher in children with elevated BLLs [median(P₂₅–P₇₅), 117.69(52.31–233.63) pg/mL] than in children with low BLLs [median(P₂₅–P₇₅): 17.70(10.75–26.52) pg/mL] (p = 0.000). Leptin was lower in children with elevated BLLs [median(P₂₅–P₇₅): 1658.23(1421.86–2606.55) pg/mL] than in children with low BLLs [median(P₂₅–P₇₅): 4168.68(3246.32–4744.94) pg/mL] (p = 0.000). In children with low BLLs, leptin was higher in children with BLLs < 3 μg/dL (N = 7) [median(P25–P75): 7220.86(4265.72–7555.15) pg/mL] than in children with BLL ≥ 3 μg/dL (N = 33) [median(P₂₅–P₇₅): 4103.86(3163.40–4678.34) pg/mL] (p = 0.026); IL-8 was significantly different in children with BLL < 4 μg/dL (N = 13) [median(P₂₅–P₇₅): 12.49(8.25–14.86) pg/mL] than in children with BLL ≥ 4 μg/dL (N = 27) [median(P₂₅–P₇₅): 21.98(13.64–33.50) pg/mL] (p = 0.013). The results defined specific changes in cytokine expressions to lead exposure, which can be used to explore the mechanism of lead toxicity and monitor lead exposure.     

A liquid chromatography–tandem mass spectrometry method for the quantification of PAC-1 in rat plasma

A sensitive and specific liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on an ACQUITY UPLC™ BEH C₁₈ column, with acetonitrile and water (39:61 (v/v) both containing 0.1% formic acid) as mobile phase at a flow rate of 0.20 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The calibration curve was linear over the range of 10–1500 ng/mL (r > 0.99). The LOQ was evaluated to be 0.3 ng/mL. The method described herein is sensitive, selective and faster than other existing method, and was successfully applied to the pharmacokinetic study and gender difference investigation of PAC-1 after oral administration in rats.     

Determination of strychnine and brucine in rat plasma using liquid chromatography electrospray ionization mass spectrometry

A simple, sensitive and selective liquid chromatography–electrospray mass spectrometric (LC–ESI-MS) method was developed and validated for simultaneous determination of strychnine and brucine in rat plasma, using tacrine as the internal standard (IS). Sample preparation involved a liquid–liquid extraction of the analytes with n-hexane, dichloromethane and isopropanol (65:30:5, v/v/v) from 0.1 mL of plasma. Chromatographic separation was carried out on a Waters C₁₈ column using a mobile phase of methanol–20 mM ammonium formate–formic acid (32:68:0.68, v/v/v). Positive selected ion monitoring mode was used for detection of strychnine, brucine and the IS at m/z 335.2, m/z 395.2 and m/z 199.2, respectively. Linearity was obtained over the concentration range of 0.5–500 ng/mL for strychnine and 0.1–100 ng/mL for brucine. The lower limit of quantification was 0.5 ng/mL and 0.1 ng/mL for strychnine and brucine, respectively. The intra- and inter-day precision for both strychnine and brucine was less than 7.74%, and accuracy ranged from −4.38% to 2.21% at all QC levels. The method has been successfully applied to a pharmacokinetic study of processed Semen Strychni after oral administration to rats.     

Fast determination of saikosaponins in Bupleurum by rapid resolution liquid chromatography with evaporative light scattering detection

A rapid resolution liquid chromatography coupled with evaporative light scattering detection method was established for simultaneous determination of six saikosaponins, namely saikosaponin a, saikosaponin c, saikosaponin d, 6″-O-acetylsaikosaponin a, 3″-O-acetylsaikosaponin d and 6″-O-acetylsaikosaponin d in Bupleurum. The analysis was performed by using an Agilent Zorbax SB-C18 column (1.8 μm, 3.0 mm × 50 mm i.d.) at gradient elution of water and acetonitrile, and the saikosaponins were well separated within 12 min, which provided about a fourfold reduction in analysis time by comparing a conventional high performance liquid chromatography method. Owing to their low ultraviolet absorption, the saikosaponins were detected by evaporative light scattering. The standard curves to quantify the saikosaponins were constructed by the log–log plot, which showed good linearity with the correlation coefficients exceeding 0.9954. The detection limits and quantification limits ranged in 8.38–25.00 μg/mL and 25.13–45.00 μg/mL, respectively. Satisfactory intra-day and inter-day precisions were achieved with the relative standard deviation (R.S.D.) less than 6.58%, and the average recoveries obtained were in the range of 96.9–100.4%. In addition, MeOH–1.0% (v/v) pyridine was found to be the best the extraction solvent when compared to MeOH and MeOH–1.0% (v/v) ammonia water. A total of 23 samples of roots of Bupleurum from different species or locations were examined with this analytical method, and their chemical profiles provided information for the chemotaxonomic investigation. The results demonstrated that the analytical method is highly effective for the quality evaluation of Bupleurum species.     

Development of a high performance liquid chromatography method for quantification of PAC-1 in rat plasma

A sensitive and specific high performance liquid chromatography method with UV detection was developed and validated for the determination of PAC-1 in rat plasma. After extraction with ethyl acetate, the chromatographic separation was carried out on a Diamonsil C₁₈ column (150 mm × 4.6 mm i.d., 5 μm particle size, Zhonghuida) protected by a ODS guard column (10 mm × 4.6 mm i.d., 5 μm particle size), using acetonitrile–methanol–phosphate buffer (pH 3.0, 30 mM) (31:3:66, v/v/v) as mobile phase at a flow rate of 1.0 mL/min, and wavelength of the UV detector was set at 281 nm. No interference from any endogenous substances was observed during the elution of PAC-1 and internal standard (IS, indapamide). The calibration curve was linear over the range of 0.05–20 μg/mL (r > 0.99). The lower limit of quantification was evaluated to be 50 ng/mL. The method was successfully applied to the pharmacokinetic study of PAC-1 after intravenous and oral administration in rats, respectively.     

Determination of underivatized amino acids to evaluate quality of beer by capillary electrophoresis with online sweeping technique

Capillary electrophoresis (CE) with ultraviolet detection was applied to determine underivatized amino acids in beer, based on the coordination interaction of copper ions and amino acids. An online sweeping technique was combined with CE to improve detection sensitivity. Using the United Nations Food Agriculture Organization/World Health Organization model of essential amino acid pattern and flavor of amino acids, the quality and taste in three kinds of beer were evaluated. It was found that Beer2 had higher quality than the other two kinds and the content of phenylalanine, proline, serine, and isoleucine was relatively large in all three kinds of beers with a great influence on beer flavor. Optimal conditions for separation were as follows: 50mM CuSO₄ at pH 4.40 as buffer; total length of fused silica capillary, 73 cm; effective length, 65 cm; separation voltage, 22.5 kV; and optimized sweeping condition, 70 seconds. In the appropriate range, linearity (r² > 0.9989), precision with a relative standard deviation < 8.05% (n = 5), limits of detection (0.13–0.25 μg/mL), limit of quantification (0.43–0.83 μg/mL), and recovery (80.5–115.8%) were measured. This method was shown to be applicable to the separation of amino acids in beer and to perform quantitative analysis directly without derivatization for the first time.     

Ultrasonic-assisted precolumn derivatization-HPLC determination of acrylamide formed in Radix Asparagi during heating process

An ultrasonic-assisted precolumn derivatization-HPLC method was established and validated for the determination of acrylamide formed in traditional Chinese herb Radix Asparagi during heating process. This method entails extraction with water, ultrasonic-assisted derivatization with 2-mercaptobenzoic acid. The final extracted acrylamide derivative was separated on C₁₈ column by using a mixture of acetonitrile and acetic acid (1 g L⁻¹ water solution) (20:80, v/v) as mobile phase. The flow rate was 0.7 mL min⁻¹ and the detection wavelength was set at 238 nm. Factors influencing the derivative reaction were evaluated and the optimum derivatization conditions were as follows: molar ratio of derivative reagent to acrylamide was 35:1, pH 8–12, ultrasonic-assisted reaction time was 100 min. The calibration curve of acrylamide showed good linearity in the range of 0.015–4.5 μg mL⁻¹ with correlation coefficient of 0.9999. The limit of detection was estimated to be 25 μg kg⁻¹ based on the signal-to-noise ratio of 3 recorded at 238 nm. Recovery of acrylamide from the sample was 106.6 ± 6.6%. Relative standard deviation of five duplicate determinations for the same sample solution was 1.59%.     

Development of a LC–MS/MS method for the determination of antrodin B and antrodin C from Antrodia camphorata extract in rat plasma for pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of antrodin B and antrodin C in rat plasma. Both target compounds, together with the internal standard (diazepam), were extracted from rat plasma samples by liquid–liquid extraction with ethyl acetate. Chromatographic separation was carried out on an Agilent XDB-C₈ column with an isocratic mobile phase consisting of acetonitrile and water (70:30, V/V) at a flow rate of 0.5 mL/min. The mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI) source operating in positive ionization mode. The assay exhibited a linear dynamic range of 47.6–4760 ng/mL for antrodin B and 56.6–5660 ng/mL for antrodin C. The intra- and inter-day precision was less than 5.3% and the accuracy was less than 2.7% for both analytes. The validated method has been applied to the pharmacokinetic study of antrodin B and antrodin C in rats following oral administration of Antrodia camphorata extract.     

Simultaneous determination of phenolic acids and flavonoids in Lycium barbarum Linnaeus by HPLC–DAD–ESI-MS

A high-performance liquid chromatography–diode array detection–mass spectrometry method with electrospray ionization mode (HPLC–DAD–ESI-MS) was developed for simultaneous determination of phenolic acids and flavonoids in fruits of Lycium barbarum Linnaeus, a widely used traditional Chinese herb possessing vital biological activity. Both phenolic acids and flavonoids were extracted with 50% ethanol and purified using a polymeric solid phase extraction cartridge followed by HPLC–DAD–ESI-MS analysis. By employing a Vydac C18 column, a total of 52 phenolic acids and flavonoids were separated within 70 min using a gradient mobile phase of 0.5% (v/v) formic acid in water and acetonitrile–water (94:6, v/v) with flow rate at 1 mL/min, column temperature at 30 °C and detection wavelength at 280 nm. Of 52 compounds, 15 phenolic acids and flavonoids were positively identified based on both absorption and mass spectra, with the remaining 37 tentatively identified by comparison of absorption spectra with reported values in the literature. Internal standards 3-hydroxybenzoic acid and hesperidin were used for quantitation of phenolic acids and flavonoids, respectively. Among the 15 positively identified compounds, quercetin-rhamno-di-hexoside was present in largest mass fraction (438.6 μg/g), followed by quercetin-3-O-rutinoside (281.3 μg/g), dicaffeoylquinic acid isomers (250.1 μg/g), chlorogenic acid (237.0 μg/g), quercetin-di-(rhamnohexoside) (117.5 μg/g), quercetin-di-(rhamno)-hexoside (116.8 μg/g), kaempferol-3-O-rutinoside (97.7 μg/g), isorhamnetin-3-O-rutinoside (72.1 μg/g), p-coumaric acid (64.0 μg/g), caffeic acid (23.7 μg/g) and vanillic acid (22.8 μg/g).     

Separation and determination of nimesulide related substances for quality control purposes by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method has been developed and validated for the determination of nimesulide related compounds in pharmaceutical formulations. Electrophoretic separation of six European Pharmacopoeia (EP) impurities (A–F) was performed using a fused silica capillary (L_eff. = 50 cm, L_tot. = 57 cm, 50 μm i.d.) with a background electrolyte (BGE) containing 25 mM borate buffer (pH 9.5), 30 mM sodium dodecyl sulphate and φ = 3% (v/v) acetonitrile. The influence of several factors (surfactant and buffer concentration, pH, organic modifier, applied voltage, capillary temperature and injection time) was studied. The method was suitably validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. The calibration curves obtained for the six compounds were linear over the range 5–12 μg ml⁻¹ (0.05–0.12%). The relative standard deviations (s_r) of intra- and inter-day experiments were less than 5.0%. The detection limits ranged between 0.7 and 1.6 μg ml⁻¹ depending on the impurity. The proposed method was applied successfully to the quantification of nimesulide impurities in its pharmaceutical formulation.