Simultaneous determination of caffeine,paracetamol, and ibuprofen in pharmaceutical formulations by high‐performance liquid chromatography with UV detection and by capillary electrophoresis with conductivity detection

Paracetamol, caffeine and ibuprofen are found in over‐the‐counter pharmaceutical formulations. In this work, we propose two new methods for simultaneous determination of paracetamol, caffeine and ibuprofen in pharmaceutical formulations. One method is based on high‐performance liquid chromatography with diode‐array detection and the other on capillary electrophoresis with capacitively coupled contactless conductivity detection. The separation by high‐performance liquid chromatography with diode‐array detection was achieved on a C₁₈ column (250×4.6 mm², 5 μm) with a gradient mobile phase comprising 20–100% acetonitrile in 40 mmol L^?1 phosphate buffer pH 7.0. The separation by capillary electrophoresis with capacitively coupled contactless conductivity detection was achieved on a fused‐silica capillary (40 cm length, 50 μm i.d.) using 10 mmol L^?1 3,4‐dimethoxycinnamate and 10 mmol L^?1 β‐alanine with pH adjustment to 10.4 with lithium hydroxide as background electrolyte. The determination of all three pharmaceuticals was carried out in 9.6 min by liquid chromatography and in 2.2 min by capillary electrophoresis. Detection limits for caffeine, paracetamol and ibuprofen were 4.4, 0.7, and 3.4 μmol L^?1 by liquid chromatography and 39, 32, and 49 μmol L^?1 by capillary electrophoresis, respectively. Recovery values for spiked samples were between 92–107% for both proposed methods.     

Efficient sample clean‐up and online preconcentration for sensitive determination of melamine in milk samples by capillary electrophoresis with contactless conductivity detection

Based on an efficient sample clean‐up and field‐amplified sample injection online preconcentration technique in capillary electrophoresis with contactless conductivity detection, a new analytical method for the sensitive determination of melamine in milk samples was established. In order to remove the complex matrix interference, which resulted in a serious problem during field‐amplified sample injection, liquid–liquid extraction was utilized. As a result, liquid–liquid extraction provides excellent sample clean‐up efficiency when ethyl acetate was used as organic extraction by adjusting the pH of the sample solution to 9.5. Both inorganic salts and biological macromolecules are effectively removed by liquid–liquid extraction. The sample clean‐up procedure, capillary electrophoresis separation parameters and field‐amplified sample injection conditions are discussed in detail. The capillary electrophoresis separation was achieved within 5 min under the following conditions: an uncoated fused‐silica capillary, 12 mM HAc + 10 mM NaAc (pH = 4.6) as running buffer, separation voltage of +13 kV, electrokinetic injection of +12 kV × 10 s. Preliminary validation of the method performance with spiked melamine provided recoveries >90%, with limits of detection and quantification of 0.015 and 0.050 mg/kg, respectively. The relative standard deviations of intra‐ and inter‐day were below 6%. This newly developed method is sensitive and cost effective, therefore, suitable for screening of melamine contamination in milk products.     

Determination of pharmaceuticals classified as emerging pollutants using capillary electrophoresis with capacitively coupled contactless conductivity detection

A study on the simultaneous separation of 13 pharmaceutical products by capillary electrophoresis with capacitively coupled contactless conductivity detection was presented. The parameters of the background electrolyte, such as pH, organic additives as well as types and concentrations of cyclodextrins (CD) were studied. The optimal separation conditions were achieved with a background electrolyte consisting of 9 mM Tris/5 mM lactic acid at pH 8.0, containing 5% n-propanol, 0.025% gamma-CD, 0.075% hydroxyl-beta-CD and 0.15% dimethyl-beta-CD. Limits of detections ranged from 61 to 1676 microg/L (S/N=3) and the relative standard deviations for migration time and peak area were below 2 and 6%, respectively. This demonstrated the potential of the capillary electrophoresis-capacitively coupled contactless conductivity detection method for biomedical and environmental analysis, as shown in the determination of pharmaceuticals identified as emerging pollutants in water samples.     

Capacitively coupled contactless conductivity detection as an alternative detection mode in CE for the analysis of kanamycin sulphate and its related substances

A method was developed to determine simultaneously kanamycin, its related substances and sulphate in kanamycin sulphate using capacitively coupled contactless conductivity detection. Kanamycin is an aminoglycoside antibiotic that lacks a strong UV-absorbing chromophore. Due to its physicochemical properties, CE in combination with capacitively coupled contactless conductivity detection was chosen. The separation method uses a BGE composed of 40 mM 2-(N-morpholino)ethanesulphonic acid monohydrate and 40 mM L-histidine, pH 6.35. A 0.6 mM N-cetyltrimethyl ammonium bromide (CTAB) solution was added as electroosmotic flow modifier in a concentration below the critical micellar concentration (CMC). Ammonium acetate 50 mg/L was used as internal standard. In total, 30 kV was applied in reverse polarity on a fused-silica capillary (65/41 cm; 75 μm id). The optimized separation was obtained in less than 6 min with good linearity (R(2)=0.9999) for kanamycin. It shows a good precision expressed as RSD on the relative peak areas equal to 0.3 and 1.1% for intra-day and inter-day precision, respectively. The LOD and LOQ are 0.7 and 2.3 mg/L, respectively. Similarly, for sulphate, a good linearity (R(2)=0.9996) and precision (RSD 0.4 and 0.6% for intra-day and inter-day, respectively) were obtained.     

Identification of inorganic ions in post‐blast explosive residues using portable CE instrumentation and capacitively coupled contactless conductivity detection

Novel CE methods have been developed on portable instrumentation adapted to accommodate a capacitively coupled contactless conductivity detector for the separation and sensitive detection of inorganic anions and cations in post‐blast explosive residues from homemade inorganic explosive devices. The methods presented combine sensitivity and speed of analysis for the wide range of inorganic ions used in this study. Separate methods were employed for the separation of anions and cations. The anion separation method utilised a low conductivity 70 mM Tris/70 mM CHES aqueous electrolyte (pH 8.6) with a 90 cm capillary coated with hexadimethrine bromide to reverse the EOF. Fifteen anions could be baseline separated in 7 min with detection limits in the range 27–240 μg/L. A selection of ten anions deemed most important in this application could be separated in 45 s on a shorter capillary (30.6 cm) using the same electrolyte. The cation separation method was performed on a 73 cm length of fused‐silica capillary using an electrolyte system composed of 10 mM histidine and 50 mM acetic acid, at pH 4.2. The addition of the complexants, 1 mM hydroxyisobutyric acid and 0.7 mM 18‐crown‐6 ether, enhanced selectivity and allowed the separation of eleven inorganic cations in under 7 min with detection limits in the range 31–240 μg/L. The developed methods were successfully field tested on post‐blast residues obtained from the controlled detonation of homemade explosive devices. Results were verified using ion chromatographic analyses of the same samples.     

Single‐run capillary electrophoresis method for the fast simultaneous determination of amoxicillin,clavulanate, and potassium

We report a new fast method for the simultaneous determination of amoxicillin, clavulanate, and potassium by capillary electrophoresis with capacitively coupled contactless conductivity detection. Samples containing potassium as the cation, and both amoxicillin and clavulanate as anions were determined simultaneously in a single run (in less than 45 s) using 10 mmol/L of both 2‐amino‐2‐hydroxymethyl‐propane‐1,3‐diol and 3‐{[2‐hydroxy‐1,1‐bis(hydroxymethyl)ethyl]amino}‐1‐propanesulfonic acid (pH 8.4) as the background electrolyte. Limits of detection were 25.0, 5.0, and 4.0 μmol/L for amoxicillin, clavulanate, and potassium, respectively. The proposed method is inexpensive, simple, fast (75 injections h⁻¹), environment friendly (minimal waste generation), and accurate (recovery values between 98 and 103%). The results obtained with the proposed method were statistically similar (95% confidence level) to those obtained by using high‐performance liquid chromatography (amoxicillin and clavulanate) and flame photometry (potassium).     

Capillary electrophoresis with capacitively coupled contactless conductivity detection method development and validation for the determination of azithromycin,clarithromycin, and clindamycin

A capillary electrophoresis with capacitively coupled contactless conductivity detection based method for the assay of azithromycin, clarithromycin and clindamycin was optimized and validated in this study. A buffer solution of 20 mM 2‐(N‐morpholino) ethane sulfonic acid, 40 mM l ‐histidine and 0.6 mM cetyltrimethylammonium bromide (pH 6.39) was used for the electrophoresis. An uncoated, bare‐fused silica capillary (total length 60 cm, effective length 32 cm, 75 μm id) was used at 25°C. The sample was injected hydrodynamically at 0.5 psi for 5 s. The electrophoresis was conducted at 30 kV in reverse polarity for 6 min with 3 and 2 min of in‐between sodium hydroxide (0.1 M) and background electrolyte rinsing, respectively. Ammonium acetate was used as internal standard. This simple and robust method showed reasonable limit of detection and limit of quantitation for azithromycin (0.0125/0.03 mg/mL), clarithromycin (0.017/0.03 mg/mL), and clindamycin (0.038/0.06 mg/mL), with good selectivity, precision both intraday (relative standard deviation ≤ 1.0%) and interday (relative standard deviation < 3.7%), linearity (R ² > 0.999) and recovery (99 – 101.7%). The method was successfully applied for the determination of azithromycin, clarithromycin and clindamycin in formulations.     

毛细管电泳/非接触式电导法分离检测手性药物氨氯地平对映体

采用毛细管电泳/电容耦合非接触式电导( CE/C4 D),以18 mmol/L柠檬酸+6 mmol/L氨水+12 mg/L羟丙基甲基纤维素(HPMC)+ 35 mmol/L羟丙基-β-环糊精(HP-β-CD)为电泳运行液,熔融石英毛细管(50μm i.d.×45 cm,leff=40 cm),正高压(+15 kV)分离...     

Rapid determination of p<Emphasis Type="Italic">K</Emphasis><Subscript>a</Subscript> values of 20 amino acids by CZE with UV and capacitively coupled contactless conductivity detections

A rapid and universal capillary zone electrophoresis (CZE) method was developed to determine the dissociation constants (pK _a) of the 20 standard proteogenic amino acids. Since some amino acids are poorly detected by UV, capacitively coupled contactless conductivity detection (C⁴D) was used as an additional detection mode. The C⁴D coupling proved to be very successful on a conventional CE-UV instrument, neither inducing supplementary analyses nor instrument modification. In order to reduce the analysis time for pK _a determination, two strategies were applied: (i) a short-end injection to reduce the effective length, and (ii) a dynamic coating procedure to generate a large electroosmotic flow (EOF), even at pH values as low as 1.5. As a result, the analysis time per amino acid was less than 2 h, using 22 optimized buffers covering a pH range from 1.5 to 12.0 at a constant ionic strength of 50 mM. pK _a values were calculated using an appropriate mathematical model describing the relationship between effective mobility and pH. The obtained pK _a values were in accordance with the literature. Figure a UV (1) and C⁴D (2) detectors placed on-line on the CE capillary. b Curve of effective mobility as a function of pH for histidine     

Separation and quantification of the urinary enantiomers of 2-hydroxyglutaric acid by capillary electrophoresis with capacitively coupled contactless conductivity detection: Application to the diagnosis of D- and L-2-hydroxyglutaric aciduria

2-hydroxyglutaric aciduria is an inherited neurometabolic disorder with two major types: D-2-hydroxyglutaric aciduria and L-2-hydroxyglutaric aciduria. An easy and fast capillary electrophoresis system combined with a capacitively coupled contactless conductivity detection method was developed for the enantioseparation and determination of D- and L-2-hydroxyglutaric acid in urine. D- and L-2-hydroxyglutaric acids were separated using vancomycin as the chiral selector. The optimal separation conditions for enantiomers were achieved by the use of a buffer containing 50 mM 4-(N-morpholino) butane sulfonic acid solution (pH 6.5), an electroosmotic flow modifier (0.001% [w/v] polybrene), and 30 mM vancomycin as chiral selector. The analysis time was 6 min under optimal conditions. The optimized and validated method was successfully implemented for quantifying D- and L-2-hydroxyglutaric aciduria in patients’ urine, without any pretreatment step. The linearity of the method was determined to be in the range of 2–100 mg/L for D- and L-2-hydroxyglutaric acid in urine. The precision (relative standard deviation%) was obtained at about 7%. For D- and L-2-hydroxyglutaric acids, the limits of detection were 0.567 and 0.497 mg/L, respectively.     

Determination of the spectrum of low molecular mass organic acids in urine by capillary electrophoresis with contactless conductivity and ultraviolet photometric detection--an efficient tool for monitoring of inborn metabolic disorders

A mixture of 29 organic acids (OAs) occurring in urine was analyzed by capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) and UV photometric detection. The optimized analytical system involved a 100 cm long polyacrylamide-coated capillary (50 μm i.d.) and the background electrolyte of 20 mM 2-morpholinoethanesulfonic acid (MES)/NaOH + 10% (v/v) methanol, pH 6.0 (pH is related to the 20 mM MES/NaOH buffer in water). The LOD values obtained by C⁴D for the OAs which do not absorb UV radiation range from 0.6 μM (oxalic acid) to 6.8 μM (pyruvic acid); those obtained by UV photometry for the remaining OAs range from 2.9 μM (5-hydroxy-3-indoleacetic acid) to 10.2 μM (uric acid). The repeatability of the procedure developed is characterized by the coefficients of variation, which vary between 0.3% (tartaric acid) and 0.6% (5-hydroxy-3-indoleacetic acid) for the migration time and between 1.3% (tartaric acid) and 3.5% (lactic acid) for the peak area. The procedure permitted quantitation of 20 OAs in a real urine sample and was applied to monitoring of the occurrence of the inborn metabolic fault of methylmalonic aciduria.     

Direct analysis of formate in human plasma,serum and whole blood by in-line coupling of microdialysis to capillary electrophoresis for rapid diagnosis of methanol poisoning

A simple method using direct injection of human blood samples and quantitative analysis of formate was developed for rapid diagnosis of methanol poisoning. A sample pretreatment device including a 500 Da molecular weight cut-off dialysis membrane was in-line coupled to capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C⁴D). 50 μL of 1:9 diluted blood samples and 50 μL of DI water were filled into the donor and the acceptor chamber, respectively, and small ionic species in blood samples were electrokinetically injected across the dialysis membrane directly into the separation capillary. Matrix components, such as red blood cells, proteins, lipids and other high molecular weight compounds, were retained by the dialysis membrane and did not interfere with subsequent CE separation. Formate was separated from other small anions in an optimized background electrolyte solution consisting of 20 mM l-histidine and 25 mM l-glutamic acid at pH 4.8. The method showed excellent analytical parameters in terms of repeatability and linearity; RSD values for migration times and peak areas at a formate concentration typical for methanol poisoning were below 0.3% and 7.4%, respectively, and linear calibration curves with correlation coefficients better than 0.999 were obtained. Limit of detection and limit of quantification were 15 and 50 μM formate in original (undiluted) blood samples, respectively. The method was applied to determination of formate in serum samples of a patient diagnosed with acute methanol poisoning.     

Dispersive micro‐solid‐phase extraction combined with online preconcentration by capillary electrophoresis for the determination of glycopyrrolate stereoisomers in rat plasma

A simple and sensitive analytical method for four isomers of glycopyrrolate in rat plasma was developed using cation‐selective exhaustive injection‐sweeping cyclodextrin‐modified electrokinetic chromatography (CSEI‐Sweeping‐CDEKC) for online enrichment combined with dispersive micro‐solid‐phase extraction pretreatment. The CSEI‐Sweeping‐CDEKC was conducted on an uncoated fused silica capillary (40.2 cm × 75 μm) with an applied voltage of –20 kV. The electrophoretic analysis was carried out in 30 mM phosphate solution at pH 2.0 containing 20 mg/mL sulfated‐β‐cyclodextrin and 5% acetonitrile. Under these optimized conditions, the detection limit for racemic glycopyrrolate was found to be 2.0 ng/mL and this method could increase 495‐fold detection sensitivity compared with the traditional injection method. Additionally, the parameters that affected the extraction efficiency of dispersive micro‐solid‐phase extraction were also examined systematically. The glycopyrrolate isomers in rat plasma samples as low as 0.0625 μg/mL were able to be separated and detected by capillary electrophoresis with the aid of CSEI‐sweeping. The findings of this study show that the dispersive micro‐solid‐phase extraction pretreatment coupled with CSEI‐Sweeping‐CDEKC is a rapid and convenient method for analyzing glycopyrrolate isomers in rat plasma.     

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

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

Enantioseparation of tartaric acid by ligand‐exchange capillary electrophoresis using contactless conductivity detection

A method for the determination of tartaric acid enantiomers using CE with contactless conductivity detection has been developed. Cu(II) as a central metal ion together with l ‐hydroxyproline were used as a chiral selector, the BGE was composed of 7 mM CuCl_2, 14 mM trans‐4‐hydroxy‐l ‐proline, and 100 mM ε‐aminocaproic acid; the pH was adjusted to 5 by hydrochloric acid. Separation with a resolution of 1.9 was achieved in 9 min in a polyacrylamide‐coated capillary to suppress the EOF. Various counterions of the BGE were studied, and migration order reversal was achieved when switching from ε‐aminocaproic acid to l ‐histidine. With detection limits of about 20 μM, the method was applied to the analysis of wine and grape samples; only l ‐tartaric acid was found.     

Heart‐cutting 2D‐CE with on‐line preconcentration for the chiral analysis of native amino acids

The use of transient moving chemical reaction boundary (tMCRB) was investigated for the on‐line preconcentration of native amino acids in heart‐cutting 2D‐CE with multiple detection points using contactless conductivity detection. The tMCRB focusing was obtained by using ammonium formate (pH 8.56) as sample matrix and acetic acid (pH 2.3) as a BGE in the first dimension of the heart‐cutting 2D‐CE. Different experimental parameters such as the injected volume and the concentration in ammonium formate were optimized for improving the sensitivity of detection. A stacked fraction from the first dimension was selected, isolated in the capillary, and then separated in the second dimension in the presence of a chiral selector ((+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid). This on‐line tMCRB preconcentration coupled with heart‐cutting 2D‐CE was applied with success to the chiral separation of D ,L ‐phenylalanine, and D ,L ‐threonine in a mixture of 22 native amino acids. The sample mixture was diluted in 0.8 M of ammonium formate, and injected at a concentration of 2.5 μM for each enantiomer with a volume corresponding to 10% of the total capillary volume. An LOD (S/N=3) of 2 μM was determined for L ‐threonine.     

Determination of oxalate and citrate in urine by capillary electrophoresis using solid‐phase extraction and capacitively coupled contactless conductivity based on an improved mini‐cell

A new method for the rapid determination of the metabolites oxalate and citrate in urine samples was based on capillary electrophoresis and capacitively coupled contactless conductivity detection coupled with solid‐phase extraction. The detection cell for capacitively coupled contactless conductivity detection was improved with a smaller inner volume (1.5 nL), reduced noise (0.2∼0.5 mV) and better reproducibility and durability. Under optimal conditions, oxalate and citrate can achieve baseline separation within 4 min and the detection limits (S/N = 3) for oxalate and citrate are about 44 and 244 ng/mL, respectively. The overall recovery is between 80.0 and 89.2%. This method offers a better choice for quantitative analysis of strong anions such as oxalate and citrate in diagnostic testing associated with human diseases.     

Capillary electrophoresis with capacitively coupled contactless conductivity detection for the determination of urinary ethylmalonic acid for the diagnosis of ethylmalonic aciduria

Ethylmalonic acid is a metabolic organic acid, and its accumulation in urine is diagnostic of ethylmalonic aciduria. In this study, a simple and fast method employing capillary electrophoresis equipped with capacitively coupled contactless conductivity detection was developed for the detection of ethylmalonic acid in urine samples. The optimized electrophoretic separation was performed in 50 mmol/L 2‐(N‐morpholino)ethanesulfonic acid solution, buffered at a pH of 6.5, and contained 0.13 mmol/L cetyltrimethylammonium bromide as an electroosmotic modifier. Electrophoresis was run at 28 kV in reversed polarity. The linear range of ethylmalonic acid concentration was between 1 and 100 mg/L with a regression coefficient of 0.9998. This method had good intra‐ and interday precision with <5% relative standard deviations. The detection limit (signal‐to‐noise ratio = 3) and the quantification limit (signal‐to‐noise ratio = 10) values were 0.139 and 0.466 mg/L, respectively. Using our optimized conditions, the method was successfully employed for the detection of ethylmalonic acid in urine sample of ethylmalonic aciduria patient.     

毛细管电泳/非接触式电导法分离检测氧氟沙星对映体

采用毛细管电泳-电容耦合非接触式电导(CE-C4D),以20 mmol/L HAc + 6 mmol/L NaAc+12 mg/L羟丙基甲基纤维素(HPMC)+35 mmol/L羟丙基-β-环糊精(HP-β-CD)为电泳运行液,在熔融石英毛细管柱(45 cm×50 μm i.d.,有效长度 40 cm)中,正高压分离,手性药物氧氟沙星对映体获得良好的基线分离,线性检测范围为0.8～40 mg/L,检出限为0.3 mg/L.考察了电泳运行液组成、二元手性选择剂(HP-β-CD和HPMC)的浓度、进样方式和样品基质等对灵敏度和分离度的影响.本方法应用于市售外消旋和左旋氧氟沙星片剂中对映体的分离测定.     

毛细管电泳-非接触式电导法直接测定偏硅酸

采用毛细管电泳-电容耦合非接触式电导检测(CE-C<'4>D),以2.4mmol/L KOH+1.6mmol/L K<,2>HPO<,4>+0.4 mmol/L 十六烷基溴化铵(CTMAB)为电泳运行液,融硅石英毛细管(45cm×50μm,有效长度40 cm),负高压分离(-15 kV),偏硅酸可在6.0 min内实现...