Potential of microchip electrophoresis in pharmaceutical analysis: Development of a universal method for frequently prescribed nonsteroidal anti-inflammatory drugs.

A novel microchip electrophoresis method with conductivity detection for the determination of nonsteroidal anti-inflammatory drugs (NSAIDs) in several pharmaceutical formulations was developed. The three frequently used NSAIDs - acetylsalicylic acid, diclofenac and ibuprofen were baseline separated on a poly(methyl methacrylate) microchip with coupled separation channels. Elimination of matrix components such as excipients, was realized through online combination of isotachophoresis (ITP) with zone electrophoresis (ZE). ITP-ZE hyphenation can also facilitate preconcentration of target analytes. ITP was carried out in the first separation channel at pH 6.5, while the second channel of the microchip was used for ZE separation and detection of the analytes at pH 7.0. The developed ITP-ZE method was demonstrated to be applicable for direct and reliable determination of NSAIDs in eleven pharmaceutical formulations. The noticeable advantage of this approach is that only simple sample pretreatment (filtration and dilution) is necessary. The method performance parameters, such as linearity (20-250% of nominal concentration of studied NSAIDs in the test samples), accuracy (98-102%) and precision (less than 2% RSD) were obtained. This universal approach is suitable for the determination of frequently used NSAIDs in a single analysis in less than 15 min. In addition to simple sample pretreatment, low running costs and minimal environmental impact could make this method attractive for the analysis of pharmaceutical preparations.

Microchip isotachophoresis coupled to surface-enhanced Raman spectroscopy for pharmaceutical analysis.

A novel online coupling of microchip isotachophoresis (µITP) with surface-enhanced Raman spectroscopy (SERS) for the analysis of complex samples is presented. Polymeric microchip with coupled channels was used for µITP-SERS analysis of four structurally similar Raman active synthetic dyes (brilliant black BN, carmoisine, ponceau 4R, and sunset yellow FCF) in pharmaceuticals. The µITP separation and simultaneous pre-concentration of the analytes were performed in the first channel of the microchip at pH 6.0 with the aid of non-Raman active discrete spacers (acetate, butyrate, glutarate, pantothenate, and valerate). Silver nanoparticles used for Raman enhancement were present in the second channel, and individual SERS spectra of the dyes were acquired by a mini Raman spectrometer operating at 532 nm. The analytical enhancement factors for silver nanoparticles were 1-5 × 104. The microchip with coupled channels enabled independent µITP separation and SERS detection, and eliminated any adverse impact of nanoparticles on the separation. The developed approach allowed reliable online SERS identification and detection of dyes with limits of detection ranging from 12 to 62 nM. Synthetic dyes were successfully separated, identified, and quantified in pharmaceutical preparations within 7 min without the need for complex or time-consuming sample pretreatment. The results were in good agreement with those obtained by an independent analytical method reported for studied dyes. Graphical abstract.

Microchip capillary electrophoresis of nitrite and nitrate in cerebrospinal fluid.

Microchip capillary electrophoresis (MCE) is a relatively new analytical method requiring only small sample amounts, which is very favorable for the analysis of volume-limited biofluids. The practical use of MCE in bioanalysis is still restricted in terms of requirements for simplifying and/or concentrating sample pretreatment techniques. Here, we describe an MCE method for trace analysis of nitrite and nitrate, indicators of various neurological diseases, in cerebrospinal fluid (CSF). The complex CSF samples were simplified by solid-phase microextraction prior to an online combination of isotachophoresis with capillary zone electrophoresis performed on a microchip with coupled channels and a high-volume sample injection channel (9.9 µL). The method is suitable for rapid (total analysis time lasted 20 min), reproducible (0.6-2.4 % RSD for migration time), and sensitive (3-9 nM limits of detection) determinations of nitrite and nitrate in 15-50 times diluted CSF samples.

Determination of metabolic organic acids in cerebrospinal fluid by microchip electrophoresis.

A new MCE method for the determination of oxalic, citric, glycolic, lactic, and 2- and 3-hydroxybutyric acids, indicators of some metabolic and neurological diseases, in cerebrospinal fluid (CSF) was developed. MCE separations were performed on a PMMA microchip with coupled channels at lower pH (5.5) to prevent proteins interference. A double charged counter-ion, BIS-TRIS propane, was very effective in resolving the studied organic acids. The limits of detection (S/N = 3) ranging from 0.1 to 1.6 µM were obtained with the aid of contact conductivity detector implemented directly on the microchip. RSDs for migration time and peak area of organic acids in artificial and CSF samples were <0.8 and <9.7%, respectively. Recoveries of organic acids in untreated CSF samples on the microchip varied from 91 to 104%. Elimination of chloride interference, a major anionic constituent of CSF, has been reached by two approaches: (i) the use of coupled channels microchip in a column switching mode when approximately 97-99% of chloride was removed electrophoretically in the first separation channel and (ii) the implementation of micro-SPE with silver-form resin prior to the MCE analysis, which selectively removed chloride from undeproteinized CSF samples.

Determination of nitrite and nitrate in cerebrospinal fluid by microchip electrophoresis with microsolid phase extraction pre-treatment.

A new method for the determination of nitrite and nitrate, indicators of various neurological diseases (meningitis, multiple sclerosis, Parkinson's disease) in cerebrospinal fluid (CSF) on an electrophoresis chip was developed. An on-line combination of isotachophoresis (ITP) with capillary electrophoresis (CE) on a poly(methylmethacrylate) chip assembled with coupled separation channels (CC) and contact conductivity detectors was employed. ITP separations performed at low pH (3.6) in the first separation channel enabled a highly selective transfer of the analytes to the second CE stage working under micellar conditions implemented by zwitterionic surfactant, 3-(N,N-dimethyldodecylammonio)-propanesulfonate. The proposed method achieved low limits of detection varied from 0.2 to 0.4µgL(-1) when the sample volume injected onto the chip (9.9µl) was almost the same as the volume of both separation channels. Preferable working conditions on the CC chip (suppressed hydrodynamic and electroosmotic flow) contributed for reproducible migration velocities (intra-day reproducibility up to 2.1% RSD) and determinations of trace concentrations of nitrite and nitrate (intra-day precision up to 3.0% RSD). Huge amount of chloride present in CSF (approx. 4.5gL(-1)) was removed from analyzed CSF samples by microsolid phase extraction performed on silver-form resin prior to the ITP-CE analysis. Developed method provided fast (approx. 20min total analysis time) and reliable determinations of trace nitrite and nitrate and could be fully integrated into the analysis of CSF samples.

CZE study on adsorption processes of aliphatic and aromatic amines on PMMA chip.

Adsorption processes on a PMMA chip linked with CZE separations of a group of 13 aliphatic and aromatic mono- and di-amines were studied. Due to the lack of chromophores within aliphatic amines, contact conductivity detection implemented directly onto the chip was used for monitoring of cationic CZE separations. To prevent an adsorption of studied amines to the chip channels, the surface of PMMA chip was modified by dynamic coating. Different surface modifiers, such as aliphatic oligoamines (diethylenetriamine and triethylenetetramine), were added to the BGE solutions filling the chip channels. The effect of various concentrations of surface modifiers on peak profiles and separation parameters of amines was monitored. Of these, mainly, aliphatic di-amines and aromatic mono-amines adversely affected the CZE resolution of a whole group of analytes by their strong adsorption to the chip channels. A propionate BGE with pH 3.2 containing 100 µM triethylenetetramine and 25 mM 18-crown-6-ether was found suitable for CZE resolution of 12 from a total of 13 amines studied. Simple dynamic modification of the surface of PMMA chip enabled fast (analysis time lasted 9 min), sensitive (sub-µM LODs reached) and reproducible (1-3% RSD of the peak areas) CZE analysis of the aliphatic and aromatic amines.