Determination of cefepime in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection

A simple micellar capillary electrokinetic chromatography (MEKC) with UV detection is described for analysis of cefepime in plasma and cerebrospinal fluid by direct injection without any sample pretreatment. The separation of cefepime from biological matrix was performed at 25 degrees C using a background electrolyte consisting of tris(hydroxymethyl)aminomethane (Tris) buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Several parameters affecting the separation of the drug were studied, including the pH and concentrations of the Tris buffer and SDS. Using cefazolin as an internal standard, the linear ranges of the method for the determination of cefepime in plasma and cerebrospinal fluid were 1-50 and 1-20 microg/mL, respectively; the detection limits of plasma (signal-to-noise ratio = 3; injection, 5 kV, 5 s) and cerebrospinal fluid (signal-to-noise ratio = 3; injection, 0.5 psi, 3 s) were 0.2 microg/mL and 0.3 microg/mL, respectively. Application of the proposed method for determination of cefepime in plasma and cerebrospinal fluid collected after intravenous administration of 2 g cefepime in patients with meningitis was demonstrated.     

Rapid determination of piracetam in human plasma and cerebrospinal fluid by micellar electrokinetic chromatography with sample direct injection

A simple micellar electrokinetic chromatography (MEKC) method with UV detection at 200 nm for analysis of piracetam in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of piracetam from biological matrix was performed at 25 degrees C using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Using imidazole as an internal standard (IS), the linear ranges of the method for the determination of piracetam in plasma and in CSF were all between 5 and 500 microg/mL; the detection limit of the drug in plasma and in CSF (signal-to-noise ratio=3; injection 0.5 psi, 5s) was 1.0 microg/mL. The applicability of the proposed method for determination of piracetam in plasma and CSF collected after intravenous administration of 3g piracetam every 6h and oral administration 1.2g every 6h in encephalopathy patients with aphasia was demonstrated.     

Rapid determination of acyclovir in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection and its clinical application

A simple MEKC with UV detection at 254 nm for analysis of acyclovir in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of acyclovir from biological matrix was performed at 25 degrees C using a BGE consisting of Tris buffer with SDS as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Using dyphylline as an internal standard, the linear ranges of the method for the determination of acyclovir in plasma and in CSF all exceeded the range of 2-50 microg/mL; the detection limit of the drug in plasma and in CSF (S/N = 3; injection 3.45 kPa, 5 s) was 1.0 microg/mL. The applicability of the proposed method for determination of acyclovir in plasma and CSF collected at 8 h after intravenous administration of 500 mg acyclovir (Zovirax) in two patients with herpes simplex encephalitis was demonstrated.     

Determination of substituted purines in body fluids by micellar electrokinetic capillary chromatography with direct sample injection.

Many substituted purines (theobromine, caffeine, paraxanthine, theophylline and uric acid, as well as other methylated xanthines and uric acids) can easily be separated and analysed in one run using micellar electrokinetic capillary chromatography with a boratephosphate buffer containing 75 mM sodium dodecyl sulphate (pH approximately 9). Serum, saliva and urine samples collected after the self-administration of caffeine and serum samples from patients receiving theophylline or caffeine pharmacotherapy were screened for substituted purines. The data presented show the ease of using on-column multi-wavelength detection for investigating the feasibility of direct sample application, the characterization of sample pretreatment procedures and peak confirmation by comparing absorption spectra. It is shown that the determination of purines in serum and saliva samples, including therapeutic concentrations of caffeine and theophylline, can be accomplished without any sample pretreatment, whereas sample extraction is required for the determination of purines in urine. Quantitative data for the determination of micromolar amounts of theophylline (samples from adult patients) and caffeine (samples from infants born prematurely) in serum samples compared well with data obtained by non-isotopic immunoassays. Micellar electrokinetic capillary chromatography with the direct injection of serum or saliva samples requires only microlitre volumes of sample and several different compounds can be determined within a few minutes.     

Separation of serum bilirubin species by micellar electrokinetic chromatography with direct sample injection.

Four major bilirubin species in serum were separated by micellar electrokinetic chromatography with 25 mM sodium dodecyl sulfate (SDS) and 20 mM sodium tetraborate-boric acid buffer at pH 8.5. Due to the solubilization of the serum proteins by the SDS micelles, serum samples were injected directly into a 50 cm x 75 microns I.D. fused-silica capillary and complete separation of the four bilirubin species was accomplished within ca. 10 min without extensive sample pretreatment. Detection was performed by absorbance at 450 nm and average limit of detection was in the 6.0 microM concentration range. The usefulness of this method was demonstrated for the separation and detection of a number of bilirubin species present in pathological human serum samples.     

Determination of procaine and tetracaine in plasma samples by micellar liquid chromatography and direct injection of sample

Summary A liquid chromatographic procedure is proposed for the determination of procaine and tetracaine in plasma samples with direct injection. The method uses a Spherisorb octadecylsilane ODS-2 C₁₈ analytical column and a micellar mobile phase containing 0.15 M sodium dodecyl sulphate, 0.5% triethylamine at pH 2.5 and 10% propanol. The UV detection was carried out at 300 nm. Plasma sample preparation required only adequate dilution with the mobile phase before injection into the chromatographic system. The proposed method allows the determination of procaine and tetracaine in plasma at therapeutic levels.     

Determination of plasma heparin by micellar electrokinetic capillary chromatography

The micellar electrokinetic capillary chromatography (MECC) method is reported for the separation of heparin, and for the possibility of direct determination of free heparin in plasma. The conditions for MECC were: pH 8.5, 25 mM sodium dodecyl sulfate (SDS), 25 mM borate buffer, with a 30 cmx50 mum ID fused-silica capillary. The sample was detected with a UV-detector at 270 nm with heparin as external standard. The recovery rate was 95.6-98.7%. This method was linear in the range 80-7000 U l(-1). The within-run and between-run relative standard deviations were lower than 3.1 and 4.5%, respectively. It is suggested that this MECC method may be used to determine blood samples containing high levels of heparin.     

场放大进样-胶束扫集毛细管电脉法测定痕量泼尼松

建立了胶束毛细管电动色谱在线富集技术测定药品中痕量的泼尼松的方法。在胶束扫集的基础上联用场放大进样,使泼尼松的富集倍数提高了136倍;检出限由原来的2.7mg/L降至20μg/L。胶束扫集毛细管电泳缓冲体系为120mmol/LSDS、10mmol/LNaH2PO4(pH2.5)10%乙腈(V/V)。分离电压-20kV,进样电压-20kV,进样时间70s,进水时间180s,检测波长250nm。同时讨论了SDS浓度、样品基质pH、进样电压、进水时间和进样时间对分离效果的影响。实验结果显示:在优化实验条件下,样品的检测仅需8min,泼尼松在0.05～10mg/L的范围内线性关系良好(r=0.998)。回收率在89.4%～106%之间,相对标准偏差在2.1%～2.6%之间,可用于各种中药制剂中泼尼松的含量测定。     

Determination of synthetic antioxidants in dairy products and dietetic supplements by micellar liquid chromatography with direct sample injection

Summary A simple and rapid HPLC method for the determination of synthetic antioxidants (propyl gallate, tert-butylhydroquinone, 2,4,5-trihydroxybutyrophenone, nordihydroguaiaretic acid, octyl gallate, 3-tert-butyl-4-hydroxyanisole and dodecyl gallate) in powdered and liquid milk, cream of milk and dietetic supplements is described. The samples are diluted or solved in a micellar solution, filtered and directly injected. The retention behavior of the antioxidants on a C18 column, with micellar mobile phases containing SDS (0.05–0.15 M), n-propanol (1–9%, v/v) and 10 mM phosphate at pH 3, has been studied by using mathematical models. Retention is predicted with errors below 3%. To optimize the mobile phase composition, a procedure which takes into account the position and shape of the peaks is applied. The optimized mobile phase, which contains 0.090 M SDS and 6.6% n-propanol, allows the separation of six antioxidants in less than 13 min. Calibration curves are linear (r>0.9998) and the limits of detection range from 0.05 to 0.3 ng antioxidant, which correspond to concentrations well below the levels allowed in foods. Repeatabilities for standards containing 5 μg mL⁻¹ ranged from 0.2 to 1.6%.     

Determination of nikethamide by micellar electrokinetic chromatography

A simple, fast, sensitive and reproducible micellar electrokinetic chromatography (MEKC)–UV method for the determination of nikethamide (NKD) in human urine and pharmaceutical formulation has been developed and validated. The method exhibits high trueness, good precision, short analysis time and low reagent consumption. NKD is an organic compound belonging to the psychoactive stimulants used as an analeptic drugs. The proposed analytical procedure consists of few steps: dilution of urine or drug in distilled water, centrifugation for 2 min (12,000 g ), separation by MEKC and ultraviolet‐absorbance detection of NKD at 260 nm. The background electrolyte used was 0.035 mol/L pH 9 borate buffer with the addition of 0.05 mol/L sodium dodecyl sulfate and 6.5% ACN. Effective separation was achieved within 5.5 min under a voltage of 21 kV (~90 μA) using a standard fused‐silica capillary (effective length 51 cm, 75 μm i.d.). The determined limit of detection for NKD in urine was 1 μmol/L (0.18 μg/mL). The calibration curve obtained for NKD in urine showed linearity in the range 4–280 μmol/L (0.71–49.90 μg/mL), with R² 0.9998. The RSD of the points of the calibration curve varied from 5.4 to 9.5%. The analytical procedure was successfully applied to analysis of pharmaceutical formulation and spiked urine samples from healthy volunteers.     

Determination of polar pesticides in soil by micellar electrokinetic chromatography using QuEChERS sample preparation

The optimal conditions for the separation of pesticides of the following classes: phenoxy carboxylic acids, sim-triazines, triazinones, chloroacetamides, urea derivatives, neonicotinoids, carbamates, triazoles, imidazoles, benzimidazoles, and phosphorus organic compounds by micellar electrokinetic chromatography have been selected. A method for determining 27 polar pesticides in soil using QuEChERS sample preparation has been developed. The recovery of pesticides was from 31 to 104%. The lower limits of the found pesticide concentrations in soil taking into account preconcentration were 0.01–0.4 mg/kg. The relative standard deviation of the results of analysis does not exceed 10%.     

Simultaneous electrokinetic and hydrodynamic injection with on-line sample concentration via micelle to solvent stacking in micellar electrokinetic chromatography

Simultaneous electrokinetic and hydrodynamic injection (SEHI) of organic cations (tricyclic antidepressant and beta blocker drugs) with on-line sample concentration using micelle to solvent stacking (MSS) was studied in micellar electrokinetic chromatography. Compared to conventional injection, >300-fold improvements in signals were obtained by hydrodynamic injection. However, with SEHI the amount of sample ions introduced into the capillary was increased which afforded a higher gain of up to 4000-fold without compromise to separation efficiency. The electrokinetic injection at negative polarity (anode at the detector end) introduced the micelle bound analytes. The hydrodynamic injection also maintained the MSS boundary inside the capillary. The stability of the MSS boundary affected SEHI where mild conditions that were low voltage as well as pressure injection were desired. The limits of detection were in the range from 0.6–4.2 ng mL⁻¹. A strategy for optimization was described and the method was applied to the ng mL⁻¹ analysis of spiked wastewater after simple dilution and centrifugation.     

Determination of itraconazole and hydroxyitraconazole in human serum and plasma by micellar electrokinetic chromatography

The electrokinetic separation of the hydrophobic antimycotic drug itraconazole (ITC) and its major metabolite, hydroxyitraconazole (HITC), by a binary aqueous-organic solvent medium containing sodium dodecylsulfate, by microemulsion electrokinetic chromatography (MEEKC) and by micellar electrokinetic chromatography (MEKC) was studied. The results suggest that the first approach is difficult to apply and that there is no substantial difference between separations performed using MEEKC and MEKC modified with n-butanol. The simpler MEKC method is more than adequate and was thus employed for the analysis of ITC and HITC in human serum and plasma. Separation was achieved in plain fused-silica capillaries having a low-pH buffer (pH 2.2) with sodium dodecyl sulfate micelles and reversed polarity. The addition of 2-propanol and n-butanol enhanced analyte solubility and altered the selectivity of the separation by influencing the magnitude of the electrophoretic component in the separation mechanism. Under optimised conditions and using head-column field-amplified sample stacking, an internal standard, ITC and two forms of HITC could be separated in under 9 min, with detection limits less than 0.01 microg/mL. Analysis of samples from patients currently prescribed ITC revealed a different HITC peak area ratio to that of the standards, suggesting a stereoselective component of ITC metabolisation. Comparison of MEKC data with those of a HPLC method employed on a routine basis showed excellent agreement, indicating the potential of this approach for therapeutic drug monitoring of ITC.     

Determination of pesticides in wine using micellar electrokinetic chromatography with UV detection and sample stacking

In this work, the analysis of a group of four fungicides (pyrimethanil, nuarimol, procymidone and cyprodinil) and one insecticide (pirimicarb) by micellar electrokinetic chromatography (MEKC) with UV detection using the on-line preconcentration strategy called reversed electrode polarity stacking mode (REPSM) is proposed. After optimisation, an adequate separation electrolyte for the separation and stacking of these pesticides was obtained which consisted of 100 mM borate, 60 mM sodium dodecyl sulphate (SDS), at pH 9.0 and 2% 2-propanol. The use of this running buffer together with the REPSM preconcentration method provided limits of detection (LODs) between 38.3 and 241 microg/L. In order to apply the developed methodology for the analysis of these pesticides in wine samples, several off-line preconcentration strategies (mainly, solid-phase extraction, SPE, and solid-phase microextraction, SPME) were tested. Although the use of a SPE procedure, optimized in this work for water samples, using Oasis HLB cartridges, provided mean recovery values between 79 and 100% for spiked water samples, it could not be applied to the extraction of these pesticides from wine samples due to high interference from the sample matrix. However, the use of a SPME procedure using polydimethylsiloxane/divynilbenzene (PDMS/DVB) fibers allowed the selective extraction of four of the five pesticides which could be perfectly determined. The final combination of the off-line SPME and on-line REPSM preconcentration strategies allowed obtaining LODs between 17.6 and 32.3 microg/L.     

On-line sample preconcentration in micellar electrokinetic chromatography by sweeping with anionic-zwitterionic mixed micelles

On-line preconcentration by sweeping in micellar electrokinetic chromatography using mixed micelles of sodium dodecyl sulfate (SDS)-SB-12 is presented. Because of their large micelle radius, they permit increased partitioning of hydrophobic analytes into the core. In addition, they also possess lower negative surface charge relative to pure SDS micelles so anionic analytes can be retained better due to decreased electrostatic repulsion. As the efficiency of sweeping is predicated on the magnitude of retention factors, these advantages translated to better focusing. As much as a 370-fold improvement in detector response, in terms of peak height, was obtained for some neutral steroids, while about a 360-fold improvement was obtained for some phenol derivatives, which were previously not amenable to sweeping by pure SDS micelles.     

Determination of heterocyclic compounds by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MECC) based on sodium cholate (NaCh) and sodium dodecyl sulphate (SDS) was developed for the determination of aromatic amino acids and heterocyclic legume constituents. The influence of temperature, voltage, micellar system, pH, zwitterion and modifier concentrations in the buffer on migration times, peak areas, resolution and number of theoretical plates was investigated. This MECC method makes possible the sensitive determination of the individual compounds with detection limits in the picomole range. Up to 300 000 theoretical plates per metre of capillary were obtained together with satisfactory linearity and repeatability of the NaCh method. The applicability of MECC to samples prepared from plant material, following a fast and simple technique of isolation, purification and group separation, is illustrated by selected examples.     

Determination of synthesized alpha-vitamin E by micellar electrokinetic chromatography

We developed a micellar electrokinetic chromatography method (MEKC) for the direct determination of the content of synthesized alpha-vitamin E. It was found that under the optimum separation conditions 7 mM borate + 14 mM phosphate + 15 mM sodium dodecyl sulfate (SDS) + 10 mM sodium cholate (NaCh) + 8% acetonitrile (pH 9.2) with UV detection wavelength at 214 nm, 16 kV constant voltage, and 26 degrees C constant temperature, alpha-vitamin E and its isomers can be baseline separated and alpha-vitamin E was quantitatively analyzed. In addition, the sample recovery, the limit of detection and the repeatability of the method were investigated. The influence of various parameters on the separation such as SDS concentration, NaCh concentration, buffer pH and acetonitrile percentage were also discussed.     

Determination of alkylphenol ethoxylates by micellar electrokinetic chromatography with bile salts

Octyl- and nonylphenol ethoxylates (OPEs and NPEs) with different numbers of ethoxy units (average values: n = 10 and N = 40 for OPEs, and n = 10 for NPEs) were separated by micellar electrokinetic chromatography under positive polarity using an 80 mM borate buffer of pH 8.5 containing sodium deoxycholate (SDC) or sodium cholate (SC). When sodium dodecyl sulfate (SDS) was added to the background electrolyte (BGE) in the absence of the bile salt, a single peak at a migration time longer than that of the EOF was obtained. Substituting the SDS by a bile salt, the homologues were resolved. At the same bile salt concentration, resolution between the homologues was higher with SDC than using SC. Optimum resolution between consecutive homologues was obtained with 50 mM SDC. In the presence of low or moderate amounts of acetonitrile or n-propanol, the background line improved significantly, whereas resolution may increase or decrease slightly. We propose a procedure for the determination of OPEs and NPEs with optimum resolution between the homologues as well as a modified procedure with improved selectivity for the single-run determination of other absorbing nonionic, cationic, and anionic (such as linear alkylbenzene sulfonates) surfactants in industrial and household cleaning products and its application to a variety of samples. The detection limit was ca. 28 microg x mL(-1) of total NPE (n = 10), and peak area repeatabilities at 50 microg x mL(-1) were 1.7% (intraday) and 5.6% (interday).     

Determination of steroids in biological samples by micellar electrokinetic chromatography

The possibility of determining eight adrenocorticotropic steroid hormones (cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, 17-hydroxyprogesterone, 11-deoxycortisol, and progesterone) by micellar electrokinetic chromatography (MEKC) using urea as an organic additive to the working electrolyte (a 25 mM phosphoric acid solution and 10 mM sodium dodecyl sulfate) was demonstrated. The use of online preconcentration (stacking and sweeping) allowed us to lower the detection limit for steroids to ～3 ng/mL. The total analysis time was 15 min.     

Determination of thyreostatics in animal feed by micellar electrokinetic chromatography

The determination of the thyreostatics 2-thiouracil, its derivatives (4-methyl-2-thiouracil, 4-propyl-2-thiouracil and 4-phenyl-2-thiouracil) and methimazole in manufactured dried animal feed by micellar electrokinetic chromatography (MEKC) is described. A 99 +/- 5% extraction yield at the 20 micrograms g-1 level (n = 8) was achieved by shaking the milled fodder with methanol-1 M NaOH (80 + 20). Aliquots of the supernatant were injected in a 75 microns x 33.5 cm uncoated silica capillary using pressure; separation was performed at 23 degrees C with 15 kV (positive polarity) in a background electrolyte (BGE) containing 40 mM sodium dihydrogenphosphate, 50 mM sodium dodecyl sulfate and 15 mM Tween 20 at pH 9. When the surfactants were added to the BGE, all the thyreostatics were well resolved and the fodder extracts showed lower backgrounds. The peaks appeared within the 2.25-5.2 min range with efficiencies in the 2.5 x 10(4)-8 x 10(4) range; methimazole appeared in the vicinity of the electroosmotic migration time. Calibration curves were linear within the studied range (20-200 micrograms ml-1, r2 > 0.998). Limits of detection in the extracts of spiked fodder samples ranged from 0.25 to 0.4 microgram ml-1, which corresponded to 0.6-1.0 microgram of drug per gram of fodder. Peak area repeatabilities were about 4% at the 20 micrograms ml-1 level.