Dispersive liquid-liquid microextraction followed by sweeping micellar electrokinetic chromatography-tandem mass spectrometry for determination of six breast cancer drugs in human plasma.

Herein, we have developed a novel method of aqueous-sample dispersive liquid-liquid microextraction (AqS-DLLME) followed by sweeping micellar electrokinetic chromatography-tandem mass spectrometry (MEKC-MS/MS) for simultaneous determination of breast cancer drugs letrozole, anastrozole, palbociclib, ribociclib, abemaciclib, and fulvestrant in human plasma. Coupling of MEKC to MS was possible due to the use of ammonium perfluorooctanoate (APFO) as a volatile surfactant. The MEKC and MS conditions were optimized to achieve a fast, sensitive, selective, and green analysis enabling full separation of the analytes within 16 min. Electrophoretic buffer was 125 mM APFO at apparent pH 10.5 in 32 % MeOH, while sheath liquid was 70 % MeOH with 0.2 % formic acid, delivered at 10 µL/min. Excellent extraction recoveries from plasma ranging from 89.4 to 104.9 % were obtained with a combination of protein precipitation and DLLME. The developed method was validated according to the ICH guidelines. Remarkable selectivity, accuracy (bias < 6.7 %), precision (RSD < 15.8 %), and stability (bias < 10.4 %) with insignificant matrix effect (RSD < 14.0 %) and no carry-over were obtained over a wide range of concentrations. Linearity with inter-day slope RSD lower than 8.7 % was demonstrated. With this method, very low concentrations could be detected after the injection of only 68.7 nL of the sample. The method was applied to plasma samples from six women currently receiving breast cancer treatment. Determined concentrations of the drugs of interest agreed with concentrations found in clinical studies, thus proving the suitability of the developed method for therapeutic drug monitoring as a superior alternative to published LC-MS methods.

Comparative Greenness Metric Estimates for Content Uniformity Testing of Anti-Cov-2, GS-5734 in Commercial Vials: Validated Micellar Electrokinetic Chromatographic Assay.

BACKGROUND: The antiviral drug GS-5734 remdesivir is a new phosphoramidate prodrug developed initially as a treatment for Ebola virus which then proved to have antiviral properties against other viruses. After clinical trials, it was the first antiviral to be approved by the U.S. Food and Drug Administration in 2020 to treat severe coronavirus (COVID-19) cases. The widespread current pandemic gave an urge to its fast production and marketing. Thus, new analytical methods must be available for its analysis in a fast and easy manner with low cost to be applicable in all laboratories. OBJECTIVE: In the current study, a green and economic micellar electrokinetic chromatographic (MEKC) method is proposed for remdesivir analysis. METHODS: A fused-silica capillary (58.5 cm × 50 µm id, 50 cm effective length) with 20 mM borate buffer (pH 9) and 25 mM sodium dodecyl sulfate was used under a positive potential of 30 kV at 25°C with detection at 245 nm. RESULTS: Remdesivir analysis was achieved in approximately 5 min. The method proved to be linear in range of 1-50 µg/mL with correlation coefficient, r > 0.999. CONCLUSION: The MEKC method proposed was applied to the analysis of remdesivir in its commercial vials. The method was validated per International Conference on Harmonization guidelines. HIGHLIGHTS: Green chemistry has been the focus of the analytical community in the past few years. This method is considered green due to its low energy and solvent consumption without sacrificing the method's sensitivity or selectivity. The method's green profile has been assessed by different greenness assessment scales to ensure the method is eco-friendly and can be used in the pharmaceutical industry.

Simultaneous separation and determination of four active ingredients in Picria fel-terrae Lour. and its preparations by micellar electrokinetic chromatography.

INTRODUCTION: Picfeltarraenins IA, IB and IV and acteoside are the four bioactive ingredients of Picria fel-terrae Lour. Their pharmacological effects include central inhibitory, cardiovascular, anti-inflammatory, anti-pyretic, analgesic, anti-bacterial, antioxidative and anti-tumor effects. OBJECTIVE: We aimed to develop an efficient micellar electrokinetic chromatography (MEKC) method modified with mixed organic solvents for the simultaneous separation and determination of the four components in Picriae Herba and its formulations. METHODS: Method optimization was carried out by investigating influences of significant factors on the separation, and this method was successfully applied for the determination of the four components in Picriae Herba and its formulations. RESULTS: The optimal running buffer was composed of 20 mM sodium tetraborate, 40 mM sodium cholate, 10% (v/v) methanol and 10% (v/v) isopropanol (pH 9.76). The separation voltage was 18 kV, the temperature was 25°C and the detection wavelength was 266 nm. Under the optimal separation conditions, the baseline separation of four components was achieved in less than 14 min. The correlation coefficients of the calibration curves were 0.9984-0.9995 for the analytes. The intraday and interday precision ranged from 1.5% to 2.5% and from 1.4% to 5.0%, respectively. Recoveries of analytes varied from 96.6% to 104.1%. CONCLUSION: The method was proved suitable for the determination of four components in Picriae Herba and its formulations. Good performance was obtained under optimal conditions, and the method provides an effective tool for the quality control of Picriae Herba and its formulations.

Sensitive analysis of doxorubicin and curcumin by micellar electromagnetic chromatography with a double wavelength excitation source.

Doxorubicin has been extensively used to treat cancers, and there are recent findings that the anticancer activities can be enhanced by curcumin. Although the two compounds have native fluorescence, they can hardly be quantified directly simultaneously using the laser-induced fluorescence (LIF) detection method. To avoid complex fluorescence derivatization and introduction of interfering components, a highly sensitive double wavelength excitation source LIF (D-W-Ex-LIF) detector composed of a 445-nm and 488-nm commercial laser diode was constructed to detect them simultaneously. Rhodamine 6G was selected as an internal standard, because its fluorescence can be excited at 445 nm and 488 nm. The native fluorescence of doxorubicin and curcumin and their resolution were enhanced by introducing mixed micelles. The optimal electrophoretic separation buffer was 10 mM borate buffer containing 20 mM Triton X-100, 5 mM sodium dodecyl sulfate, and 30% (v/v) methanol at pH 9.00. Therefore, the developed method was specific, accurate, and easily operable. Its limits of detection for doxorubicin and curcumin in human urine samples were 4.00 × 10-3 and 1.00 × 10-2 µg/mL, respectively, and the limits of quantification were 1.00 × 10-2 and 3.00 × 10-2 µg/mL, respectively. The recoveries were 94.9-109.1%. Graphical abstract.

Application of MEKC and UPLC with Fluorescence Detection for Simultaneous Determination of Amlodipine Besylate and Bisoprolol Fumarate.

OBJECTIVE: Two chromatographic methods were described for simultaneous determination of the antihypertensive drugs amlodipine besylate (AML) and bisoprolol fumarate (BIS). METHODS: Method I applies micellar electrokinetic capillary chromatography using a deactivated fused silica capillary (25 cm effective length × 50 µm internal diameter). The background electrolyte consisted of 0.01 M borate buffer (pH 9.2) containing 0.025 M sodium dodecyl sulphate and methanol in the ratio of 80:20 (v/v). Valsartan (VAL) was used as an internal standard. Diode array detector was set at 238, 224, and 210 nm for measuring AML, BIS, and VAL, respectively. Method II involves using ultra-performance liquid chromatography with fluorescence detection. Zorbax SB-C8 column (2.1 × 100 mm, 1.8 µm particle size) was used with isocratic elution of the mobile phase composed of 0.1% trifluoroacetic acid, acetonitrile, and methanol in the ratio of 55:35:10 (v/v) at a flow rate of 0.6 mL/min. Fluorescence detection was done using excitation wavelengths 230 and 370 nm and emission wavelengths 305 and 450 nm for BIS and AML, respectively. Validation parameters were carefully studied including linearity, ranges, precision, accuracy, robustness, detection, and quantification limits. RESULTS: Method I showed good linearity over the range 10-100 µg/mL for both dugs. Method II's linear ranges were 0.001-0.1 and 0.02-1 µg/mL for BIS and AML, respectively. CONCLUSION: The proposed methods were successfully validated and applied for assay of the studied drugs in their fixed-dose combination tablets. HIGHLIGHTS: To the best of our knowledge, this study suggests the first electro-chromatographic and LC with fluorescence detection methods for simultaneous determination of amlodipine and bisoprolol.

A simple and efficient sequential electrokinetic and hydrodynamic injections in micellar electrokinetic chromatography method for quantification of anticancer drug 5-fluorouracil and its metabolite in human plasma.

A simple and sensitive preconcentration strategy using sequential electrokinetic and hydrodynamic injection modes in micellar electrokinetic chromatography with diode array detection was developed and applied for the separation and determination of anticancer agent, 5-fluorouracil and its metabolite, 5-fluoro-2'-deoxyuridine, in human plasma. Sequential injection modes with increased analyte loading capacity using the anionic pseudo-stationary phase facilitated collection of the dispersed neutral and charged analytes into narrow zones and improved sensitivity. Several important parameters affecting sample enrichment performance were evaluated and optimized in this study. Under the optimized experimental conditions, 614- and 643-fold and 782- and 803-fold sensitivity improvement were obtained for 5-fluorouracil and its metabolite when compared with normal hydrodynamic and electrokinetic injection, respectively. The method has good linearity (1-1,000 ng/ml) with acceptable coefficient of determination (r2 > 0.993), low limits of detection (0.11-0.14 ng/ml) and satisfactory analyte relative recovery (97.4-99.7%) with relative standard deviations of 4.6-9.3% (n = 6). Validation results as well as the application to analysis of human plasma samples from cancer patients demonstrate the applicability of the proposed method to clinical studies.

[Application of micellar electrokinetic capillary chromatography for identification, quantitative detection and unfolding analysis of interleukin-12].

OBJECTIVE: To establish a micellar electrokinetic capillary chromatography-based method for identification and quantitative detection of interleukin-12 (IL-12) and analysis of its unfolding process. METHODS: An uncoated fused-silica capillary (inner diameter 50 µm) with a total length of 48.5 cm (40 cm to the detector) was used for the experiment. The factors influencing the separation efficiency of IL-12 were analyzed, and a standard curve of IL-12 concentration was established. The mixture of IL-12 and anti-IL-12 antibody was incubated in a water bath at 38 ℃ for 40 min, and capillary electrophoresis was then performed under the same conditions. The results were compared with those of IL-12 and anti-IL-12 antibody to identify IL-12. IL-12 and dithiothreitol (DTT) were incubated at 60 ℃ in water bath for different lengths of times, and the unfolding process of IL-12 was analyzed based on electrophoresis results of IL-12 in different states. RESULTS: A micellar capillary electrophoresis on-line sweep method was established with 80 mmol/L borate (pH=9.3) containing 30 mmol/L sodium dodecyl sulfate (SDS) as the buffer solution. This system showed a good linear relationship between the peak area and the mass concentration of IL-12 with a linear correlation coefficient of 0.9991 within the linear range of 2 to 120 ng/L. As the incubation time of IL-12 and DTT prolonged, the disulfide bond of IL-12 gradually opened and resulted in distinct changes in the protein peak. CONCLUSIONS: This capillary electrophoresis-based method is simple and sensitive for IL-2 analysis and allows rapid detection of changes in IL-12 content in the setting of tumors and analysis of the possible causes.

Magnetic solid-phase extraction of four ß-lactams using polypyrrole-coated magnetic nanoparticles from water samples by micellar electrokinetic capillary chromatography analysis.

In the present research, the application of a polypyrrole coating on Fe3O4 magnetic nanoparticles (PPy/Fe3O4 MNPs) as a sorbent was confirmed. The synthesized magnetic composites were characterized by TEM, FTIR spectroscopy, and XRD. Four ß-lactams (oxacillin (OXA), cloxacillin (CLOX), dicloxacillin (DIC), and flucloxacillin (FLU)) were selected as analytes for the experiment. The extracted ß-lactams were determined by micellar electrokinetic capillary chromatography-diode array detector (MEKC-DAD). The crucial parameters influencing the extraction efficiency and separation were studied and optimized. Under the optimal conditions, the limits of detection are 1.0 µg L-1 for OXA, CLOX, FLU, and 0.8 µg L-1 for DIC. The calibration curves are linear in the range of 2.5-200.0 µg L-1. The proposed method was applied for the determination of the ß-lactams in water samples with satisfactory results. The intra-day relative standard deviations and the inter-day relative standard deviations range from 1.09% to 4.58% and from 2.95% to 7.80%, respectively. It can be concluded that this method is sensitive, convenient, and feasible for the determination of the ß-lactams in water samples.

Simultaneous separation and determination of hirsutine and hirsuteine by cyclodextrin-modified micellar electrokinetic capillary chromatography.

INTRODUCTION: Hirsutine and hirsuteine are the main pharmacological activity ingredients of Uncaria rhynchophylla (UR), playing an important role in treating mental and cardiovascular diseases, such as Alzheimer's disease, hypertension, Parkinson's disease, potential anti-cancer activities and so on. OBJECTIVE: To develop a cyclodextrin-modified micellar electrokinetic capillary chromatography (CD-MEKC) method for the simultaneous separation and determination of hirsutine and hirsuteine from UR and its formulations. METHODOLOGY: The optimal method was developed by investigating influences of significant factors on the separation, and this method was successfully applied for the determination of hirsutine and hirsuteine in UR and its formulations. RESULTS: The optimal background electrolyte (BGE) consisted of 40 mM sodium dihydrogen phosphate (pH 7.0), 150 mM 2,6-dimethyl-ß-cyclodextrin (DM-ß-CD), 3 mM mono-(6-ethylenediamine-6-deoxy)-ß-cyclodextrin (ED-ß-CD), and 30 mM sodium cholate (SC). Under these conditions, hirsutine and hirsuteine were successfully separated within 13 min at the separation voltage of 15 kV, temperature of 25°C and the detection wavelength of 224 nm. For the analytes, linear calibration curves were performed within the range 5.0-160.0 µg/mL. The limit of detection (LOD, S/N = 3) and the limit of quantitation (LOQ, S/N = 10) were 0.41, 1.42 µg/mL for hirsutine and 0.60, 2.17 µg/mL for hirsuteine, respectively. The recoveries of three samples were from 97.9% to 102.3%. CONCLUSION: The method was successfully applied to the determination of hirsutine and hirsuteine in UR and its formulations. Meanwhile, it provides an effective reference of the quality control of UR and its formulations.

Determination of S-adenosylmethionine, S-adenosylhomocysteine, and methylthioadenosine in urine using solvent-modified micellar electrokinetic chromatography.

A new approach for direct determination of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and methylthioadenosine (MTA) in urine was developed based on MEKC by using SDS modified with isobutanol in the presence of PEG-300. Analytes were first extracted with grafted phenylborononic acid. Using a 50 µm internal diameter silica capillary of 32 cm total length filled with 0.05 M SDS, 0.05 M H3 PO4 , 5% (v/v) isobutanol, and 10% (v/v) PEG-300, LOQ of 0.15 µM for SAM and SAH, and 0.2 µM for MTA was reached. Accuracy was 92% for MTA, 109% for SAH, and 105% for SAM, intra- and interday imprecision were <2.5 and ≤3%, respectively. The total time of analysis for one sample was 10 min. Analysis of 30 urine samples from healthy volunteers showed that the median SAM and SAH levels were 12.1 and 0.73 µM, respectively. MTA levels, which were determined in urine for the first time (according to our data), were 0.43 µM, and these values correlated well with the SAM level (r = 0.748, p < 0.01).

Determination of phenylenediamines in hair colors derivatizated with 5-(4, 6-dichlorotriazinyl)aminofluorescein via micellar electrokinetic chromatography.

Phenylenediamines (PDs), which are reported to cause allergic dermatitis and possess genotoxicity and carcinogenicity, are the ingredients used in permanent hair dyes. The fluorescent derivatization strategy coupled with micellar electrokinetic chromatography (MEKC) were established to analyze four PDs, including o-phenylenediamine (OPD), m-phenylenediamine (MPD), p-phenylenediamine (PPD) and toluene-2,5-diamine (PTD). Additionally, 5-(4, 6-dichlorotriazinyl) aminofluorescein (DTAF) was used as a fluorescent reagent derived at amino groups of PDs and underwent nucleophilic substitution reaction to improve the detection sensitivity. The derivatization condition reacted at 90 °C for 10 min in alkaline conditions. The optimized separation conditions were 20 mM borate (pH 8.0) containing 10 mM Brij 35 and 35% (v/v) methanol. The limits of detection (S/N = 3) for MPD, PTD, PPD and OPD were 25, 25, 50 and 100 nM, respectively. Compared to MEKC-UV, the sensitivity enhancements were 30- to 81-fold when PDs were derived with DTAF. The high-sensitivity MEKC-LIF method was successfully established and applied to determine PDs in commercial hair colors for quality control and in real hair samples for evaluating the location of PDs in dyed hair samples, as well as in percutaneous absorption samples for evaluating the ability of PDs to penetrate skin.

Insulin: a review of analytical methods.

Insulin is an important polypeptide hormone that regulates carbohydrate metabolism. Abnormal levels of insulin are associated with diabetes mellitus that is characterized by chronic hyperglycemia. Therefore, reliable quantification of insulin is important for clinical purposes such as the diagnosis and treatment of diabetes. Nowadays, immunoassays and chromatographic assays are the primary methods that have been developed for insulin analysis. In this review, we have summarized the principles, sensitivity, linearity range, and recovery as well as the advantages and limitations of the most prevalent methods. Generally, immunoassays including enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), radioimmunoassay (RIA) and on-chip immunoassay show good selectivity towards insulin with less interferences and high throughput. Chromatographic assays including high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV), micellar electrokinetic capillary chromatography (MECC), and liquid chromatography with tandem mass spectrometry (LC-MS/MS) are highly sensitive and capable of simultaneous detection of insulin and its analogues.

Field-amplified sample injection and sweeping micellar electrokinetic chromatography in analysis of glyphosate and aminomethylphosphonic acid in wheat.

Glyphosate, a widely used herbicide, has been classified as probably carcinogenic to humans by the International Agency for Research on Cancer (IARC). In the present study a method based on Field-Amplified Sample Injection and Sweeping Micellar Electrokinetic Chromatography (FASI sweep-MEKC) has been developed and validated for determination of glyphosate and its microbial metabolite aminomethylphosphonic acid (AMPA) in wheat flour. The method involved a preliminary solid phase extraction for cleanup of the aqueous extracts from wheat flour, based sequentially on C18 and strong anion exchange cartridges, followed by derivatization using 9-fluorenylmethylchloroformate. Optimization of sample cleanup and derivatization procedure was carried out by a HPLC-UV method, whereas FASI sweep-MEKC was applied for achieving the sensitivity necessary for analysis of real samples. To this regard, optimum conditions involved the use of an extended path fused-silica capillary (80 cm total length, 50 µm, i.d.) filled with a high concentration buffer (sodium phosphate 100 mM, pH 2.2). Electrokinetic sampling was carried out at -10 kV with injection time of 700 s and the separation of the loaded analytes was performed under MEKC conditions using sodium phosphate buffer 50 mM at pH 2.2, supplemented with sodium dodecyl sulfate, 100 mM. The method was validated for linearity, precision, accuracy and sensitivity, showing that using conventional UV detection (210 nm) the achieved limit of quantitation (LOQ) values for both the analytes were widely lower than those set by Authorities. In particular, LOQ for glyphosate and AMPA were found to be 5 and 2.5 ng/mL, respectively, corresponding to 0.1 and 0.05 mg/kg, in wheat flour. The method, applied to commercially available real samples (wheat flour from different manufacturers) and to an experimental sample obtained by cv. Svevo wheat, can be considered as a convenient alternative to the existing approaches in analysis of complex matrices.

Application of SPME supported by ionic liquids for the determination of biogenic amines by MEKC in clinical practice.

The analysis of biogenic amines (BAs) and their metabolites is helpful for the diagnosis of central nervous system disorders and other neuroendocrine and cancer disturbances. In the study, a developed micellar electrokinetic chromatography method, coupled with diode array detection (MEKC-DAD), was validated to monitor levels of adrenaline (A), noradrenaline (NA), dopamine (DA), L-Tryptophan (L-Tryp) and L-Tyrosine (L-Tyr) in real human urine samples. These neurotransmitters were isolated from urine samples using solid-phase microextraction (SPME) and methanol containing 1-ethyl-3-methylimidazolium tetrafluoroborate ionic liquid as the desorption phase. The method was linear for DA, A and L-Tyr in the range of 0.5-20 µg/mL and for NA and L-Tryp in the range of 0.25-20 µg/mL. The good linearity for BAs was confirmed by the correlation coefficient (R2) from 0.9989 for A to 0.9997 for NA and L-Tryp, respectively. The validation assays for accuracy, precision, limit of detection, limit of quantification, absolute recovery, and stability of the analytes were consistent with the requirements recommended by the FDA and ICH guidelines. Next, the validated SPME-MEKC method was successfully used for the quantification of A, NA, DA, L-Tryp and L-Tyr in real human urine samples collected from pediatric patients suffering from neuroblastoma, ganglioneuroblastoma, Wilms' tumor, rhabdoid tumor and lipoblastomatosis, as well as from healthy volunteers. Finally, the levels of BAs in cancer patients were evaluated as to whether they can be used as biomarkers of various health disturbances.

Rapid Separation of Organophosphate Pesticides using Micellar Electrokinetic Chromatography and Short-end Injection.

Organophosphate (OP) pesticides are highly toxic substances and are frequently represented as poisons. In order to qualify and quantify the selected OP pesticides (methyl paraoxon, ethyl paraoxon, methyl parathion, fenitrothion, and ethyl parathion), micellar electrokinetic chromatography and short-end injection were investigated. This is the first time that this combination has been used to separate OP pesticides. A capillary with 8.5 cm effective length was used, and the analytes were separated within 2.1 min. Separation conditions including buffer (type, pH, and concentration), sodium dodecyl sulfate concentration, and separation voltage were optimized. The limit of detection (LOD) was estimated in the range of 10-20 µM. The OP pesticides spiked in artificial saliva and drinking water gave superior peak profiles, and good average recoveries 95.6% and 62.3%, respectively. Overall, a rapid method with excellent resolution and efficiency was developed and successfully applied in the analysis of potential sample matrixes.

[Determination of four phenolic estrogens in water samples by pressure-assisted electrokinetic injection coupled with capillary electrophoresis].

A capillary electrophoresis (CE) method was developed for the simultaneous separation and determination of four phenolic estrogens (PEs), in which pressure-assisted electrokinetic injection (PAEKI) was adopted as the on-line concentration method to enrich the PEs. Several parameters affecting PAEKI conditions such as injection voltage and injection time, were systematically investigated and compared with the usual parameters. Under the optimized PAEKI conditions (-9 kV, 0.3 psi (ca. 2.1 kPa), 0.4 min), the four PEs separated completely within 7 min. Good linearities were attained in the range of 0.05-5 mg/L for hexestrol and dienestrol, and 0.1-10 mg/L for bisphenol A and diethylstilbestrol, with correlation coefficients (r) over 0.9936. The limits of detection (S/N=3) were 0.0071-0.017 mg/L, and enrichment factors ranged from 11 to 15 compared to normal hydrodynamic injection. The combined micellar electrokinetic chromatographic-PAEKI method developed was used to determine the PEs in tap water and lake water samples; limits of quantification (S/N=10) of 0.029-0.064 mg/L and 0.033-0.079 mg/L were attained, respectively, by the two sample types. Furthermore, recoveries ranged from 75.6% to 110.1% with relative standard deviations (n=5) within 4.6%-11.8%. To use this PAEKI method, researchers would only need to adjust the parameters of the CE apparatus to perform on-line enrichment of analytes, without using other reagents; this demonstrates the simplicity, rapidity, and highly automated nature of this method.

Chiral analysis of theanine and catechin in characterization of green tea by cyclodextrin-modified micellar electrokinetic chromatography and high performance liquid chromatography.

Monomeric catechins are important compounds in green tea accounting for potential bioactivity against a wide range of diseases. Besides catechins, l-Theanine (γ-glutamylethylamide), a characteristic amino acid in tea leaves, has become a further focus of the phytochemical research for the reported beneficial effects mainly on cognitive performance, emotional state and sleep quality. In the present study has been developed a CD-MEKC method based on sodium dodecyl sulfate (SDS) and Heptakis (2,6-di-O-methyl)-ß-cyclodextrin for the separation of six major green tea catechins and enantiomers of theanine. The latter, because of the poor detectability was derivatized prior analysis by o-phthaldialdehyde in the presence of N-acetyl-l-cysteine which, under mild conditions (neutral pH, in two minutes) allowed two diastereomers isoindole derivatives to be obtained. The derivatization reaction was directly carried out on tea infusion and derivatized samples were analysed by CD-MEKC involving 65 mM SDS and 28 mM cyclodextrin in acidic buffer (pH 2.5). The separation of six major green tea catechins including enantioresolution of (±)-Catechin and d/l-Theanine was obtained in about 5 min allowing d-Theanine to be quantified at least at 0.5% m/m level with respect to l-Theanine. Since (-)-Catechin and d-Theanine can be considered as non-native enantiomers (distomers), their presence in real samples provides an indication of tea leaves treatments (thermal treatment, fermentation, etc.) and could represent an opportunity for grading tea. The obtained results were confirmed by a RP-HPLC approach; even though the chromatography was developed in achiral conditions, the derivatization approach applied to theanine (diastereomers formation), allowed for d/l-Theanine chiral analysis.

Simultaneous separation and determination of praeruptorin A, B and C by micellar electrokinetic chromatography using sodium dodecyl sulphate and sodium cholate as mixed micelles.

INTRODUCTION: Praeruptorin A, B and C are major bioactive constituents in Peucedani Radix. They display anti-inflammatory effect, anti-hypertension effect, antiplatelet aggregation, potential anti-cancer activities and so on. They are worthy of investigation as potentially novel and versatile drugs. OBJECTIVE: To develop a method using micellar electrokinetic chromatography (MEKC) for the application in simultaneously separation and determination of praeruptorin A, B and C from Peucedani Radix and its medicinal preparations. METHODS: Method optimisation was carried out by investigating influences of significant factors on the separation. The method was subjected to validation. The determination of praeruptorin A, B and C in Peucedani Radix and its drug formulations was accomplished by the developed method. RESULTS: The optimal separation condition was 20 mM borate buffer containing 40 mM sodium cholate (SC), 22 mM sodium dodecyl sulphate (SDS) and 25% (v/v) acetonitrile (pH 10.00); 15 kV of voltage; 25°C of temperature; detection at 224 nm. Under this condition, three analytes were baseline separated within 16 min. A good linearity was obtained with correlation coefficients from 0.9988 to 0.9995. The limits of detection (LODs) and limits of quantitation (LOQs) ranged from 0.50 to 0.80 µg/mL and from 1.50 to 2.50 µg/mL, respectively. The recoveries ranged between 95.3% and 103.4%. CONCLUSION: The proposed method has been successfully applied to the simultaneous determination of praeruptorin A, B and C in Peucedani Radix and its pharmaceutical preparations. Additionally, it could be a potential alternative to the quality control of Peucedani Radix.

Estudo da separação de glicocorticoides e aplicações em formulações farmacêuticas utilizando eletroforese capilar / Study of the separation of glucocorticoids and application in pharmaceutical formulations using capillary electrophoresis

Estudos envolvendo os glicocorticoides merecem destaque devido a serem hormônios responsáveis pela transferência de informações e instruções às células, desta forma regulando o metabolismo, desenvolvimento, crescimento, função imune e também auxiliam no controle das funções tanto reprodutivas quanto tecidual. Estes também são sintetizados e amplamente utilizados com finalidade terapêutica processos alérgicos, tratamento de doenças autoimunes, em transplantes no pré-operatório e/ou pós-operatório-, devido a sua eficiente ação como imunossupressores e anti-inflamatórios. Os dois primeiros capítulos deste trabalho exibem uma revisão da literatura com foco em considerações gerais sobre os glicocorticoides, metodologias empregadas na análise destes hormônios e fundamentos da eletroforese capilar. Na sequência, o quarto capitulo, mostra a otimização da separação de 17 glicocorticoides utilizando cromatografia eletrocinética micelar devido a alto grau hidrofóbico dos analitos. Para tal, a composição do eletrólito consistiu em 20mM de tetraborato de sódio (pH=9.3) e 30 mM de dodecil sulfato de sódio (como surfactante), e a interação soluto-micela e, portanto, retenção do soluto, foi manipulada com a adição (volume/volume) de solventes orgânicos na composição de até 20% acetonitrila (ACN), 20% etanol (EtOH) e 1% tetrahidrofurano (THF), a qual se baseia num modelo de desenho de misturas (totalizando dez diferentes eletrólitos), e através desta abordagem um ótimo de separação foi obtido (13,3% EtOH, 3,3% ACN e 0,17% THF). A melhor condição de separação foi testada qualitativamente numa amostra de urina de um voluntário que faz uso contínuo de prednisona como terapia corticoidal. As misturas de solventes estudadas neste trabalho afetam a solubilidade dos hormônios na fase aquosa e a estrutura micelar também sofre grande impacto,principalmente na camada de solvatação. O quarto capítulo busca racionalizar tais efeitos através da obtenção de descritores, e as informações contidas nos descritores hidrofóbicos e hidrofílicos são sempre relevantes e contribuem nas correlações encontradas. Obteve três grupos de comportamento distinto, onde a capacidade doadora e aceptora de prótons para a realização de ligações de hidrogênios foram as interações consideradas as mais relevantes para o comportamento observado da separação. E o capítulo final, apresenta possibilidades de aproveitamento no controle de qualidade na indústria farmacêutica, métodos baseados na injeção e tensão inversas foram propostos a fim de ganho de tempo de análise (máximo de 5 minutos), estes foram validados seguindo o protocolo preconizado pela ANVISA (Agência Nacional de Vigilância Sanitária) nos parâmetros: precisão, exatidão, seletividade, linearidade, limites de detecção e quantificação e robustez; e aplicados na quantificação de quatro (diferentes formulações comerciais contendo glicocorticoides (prednisona 20 mg, betametasona 4 mg, furoato de mometasona 200 mcg e dipropionato de beclometasona 200 mcg)

Studies involving glucocorticoids deserve to be highlighted because they are hormones responsible for the transfer of information and instructions to cells, thus regulating metabolism, development, growth, immune function and also assist in the control of both reproductive and tissue functions. These are also synthesized and widely used for therapeutic purposes - allergic processes, treatment of autoimmune diseases, in preoperative and/or postoperative transplants - due to their efficient action as immunosuppressants and anti-inflammatories. The first two chapters of this paper present a review of the literature focusing on general considerations about glucocorticoids, methodologies used in the analysis of these hormones and fundamentals of capillary electrophoresis. Subsequently, the fourth chapter shows the optimization of the separation of 17 glucocorticoids using micellar electrokinetic chromatography due to the high hydrophobic degree of the analytes. To this end, the electrolyte composition consisted of 20 mM sodium tetraborate (pH = 9.3) and 30 mM sodium dodecyl sulfate (as a surfactant), and the solute-micelle interaction and therefore solute retention was manipulated with organic solvent in the composition of up to 20% acetonitrile (ACN), 20% ethanol (EtOH) and 1% tetrahydrofuran (THF), which is based on a mixture design model (totaling ten different electrolytes), and through this approach an optimal separation was obtained (13.3% EtOH, 3.3% ACN and 0.17% THF). The best separation condition was qualitatively tested in a urine sample from a volunteer who makes continuous use of prednisone as corticosteroid therapy. The solvent mixtures studied in this work affect the solubility of the hormones in the aqueous phase and the micellar structure also has a great impact, especially on the solvation layer. The fourth chapter seeks to rationalize these effects by obtainingdescriptors, and the information contained in the hydrophobic and hydrophilic descriptors is always relevant and contributes to the correlations found. It obtained three groups of distinct behavior, where the donor and acceptor capacity of protons for the realization of hydrogen bonds were the interactions considered the most relevant for the observed behavior of the separation. And the final chapter presents possibilities of use in quality control in the pharmaceutical industry, methods based on injection and reverse voltage were proposed in order to gain analysis time (maximum of 5 minutes), these were validated following the protocol recommended by ANVISA (Brazilian National Agency of Sanitary Surveillance) in the parameters: precision, accuracy, selectivity, linearity, limits of detection and quantification and robustness; and applied in the quantification of four different commercial formulations containing glucocorticoids (prednisone 20 mg, betamethasone 4 mg, mometasone furoate 200 mcg and beclomethasone dipropionate 200 mcg)

Separation and determination of peptide metabolite of Bacillus licheniformis in a microbial fuel cell by high-speed capillary micellar electrokinetic chromatography.

A method using high-speed capillary micellar electrokinetic chromatography and a microbial fuel cell was applied to determine the metabolite of the peptides released by Bacillus licheniformis. Two peptides, l-carnosine and l-alanyl-l-glutamine were used as the substrate to feed Bacillus licheniformis in a microbial fuel cell. The metabolism process of the bacterium was monitored by analyzing the voltage outputs of the microbial fuel cell. A home-made spontaneous injection device was applied to perform high-speed capillary micellar electrokinetic chromatography. Under the optimized conditions, tryptophan, glycine, valine, tyrosine and the two peptides could be rapidly separated within 2.5 min with micellar electrokinetic chromatography mode. Then the method was applied to analyze the solutions sampled from the microbial fuel cell. After 92 h running, valine, as the metabolite, was successfully detected with concentration 3.90 × 10-5 M. The results demonstrated that Bacillus licheniformis could convert l-carnosine and l-alanyl-l-glutamine into valine. The method employed in this work was proved to have great potential in analysis of metabolites, such as amino acids, for microorganisms.