Internal standardization electrophoresis

Ohnesorge, J., Saenger-van de Griend, C., and Waetzig, H. (2005). Quantification in capillary electrophoresis-mass spectrometry long- and short-term variance components and their compensation using internal standards. Electrophoresis 26, 2360—2375. 

Linder V, Verpoorte E, de Rooij NF, Sigrist H, Thormann W. Application of surface biopassivated disposable poly(dimethylsiloxane)/glass chips to a heterogeneous competitive human serum immunoglobulin G immunoassay with incorporated internal standard. Electrophoresis 2002 23 740-749. 

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Xue, G., Pang H.-M., and Yeung E.S., Multiplexed capillary zone electrophoresis and micellar electrokinetic chromatography with internal standardization, Anal. Chem. 71, 2642, 1999. 

Gotti et al. [42] reported an analytical study of penicillamine in pharmaceuticals by capillary zone electrophoresis. Dispersions of the drug (0.4 mg/mL for the determination of (/q-penicillaminc in water containing 0.03% of the internal standard, S -met hy I - r-cystei ne, were injected at 5 kPa for 10 seconds into the capillary (48.5 cm x 50 pm i.d., 40 cm to detector). Electrophoresis was carried out at 15 °C and 30 kV, with a pH 2.5 buffer of 50 mM potassium phosphate and detection at 200 rnn. Calibration graphs were linear for 0.2-0.6 pg/mL (detection limit = 90 pM). For a more sensitive determination of penicillamine, or for the separation of its enantiomers, a derivative was prepared. Solutions (0.5 mL, final concentration 20 pg/mL) in 10 mM phosphate buffer (pH 8) were mixed with 1 mL of methanolic 0.015% 1,1 -[ethylidenebis-(sulfonyl)]bis-benzene and, after 2 min, with 0.5 mL of pH 2.5 phosphate buffer. An internal standard (0.03% tryptophan, 0.15 mL) was added and aliquots were injected. With the same pH 2.5 buffer and detection at 220 nm, calibration graphs were linear for 9.3-37.2 pg/mL, with a detection limit of 2.5 pM. For the determination of small amounts of (L)-penicillamine impurity, the final analyte concentration was 75 pg/mL, the pH 2.5 buffer contained 5 mM beta-cyclodextrin and 30 mM (+)-camphor-10-sulfonic acid, with a voltage of 20 kV, and detection at 220 nm. Calibration graphs were linear for 0.5-2% of the toxic (L)-enantiomer, with a detection limit of 0.3%. 

Fan et al. [106] developed a high performance capillary electrophoresis method for the analysis of primaquine and its trifluoroacetyl derivative. The method is based on the mode of capillary-zone electrophoresis in the Bio-Rad HPE-100 capillary electrophoresis system effects of some factors in the electrophoretic conditions on the separation of primaquine and trifluoroacetyl primaquine were studied. Methyl ephedrine was used as the internal standard and the detection was carried out at 210 nm. A linear relationship was obtained between the ratio of peak area of sample and internal standard and corresponding concentration of sample. The relative standard deviations of migration time and the ratio of peak area of within-day and between-day for replicate injections were <0.6% and 5.0%, respectively. 

Jamali, B., and Nielsen, H. M. (2003). Development and validation of a capillary electrophoresis-indirect photometric detection method for the determination of the non-UV-absorbing 1,4-dideoxy-l,4-imino-d-arabinitol in active pharmaceutical ingredients, solutions and tablets using an internal standard. J. Chromatogr. A 996(1—2), 213-223. 

Morgan NY, Wellner E, Talbot T, Smith PD, Phillips TM. Development of a two-color laser finorescence detector—On-line detection of internal standards and nnknowns by capillary electrophoresis within the same sample. Journal of Chromatography A 1105, 213-219, 2006. 

Hoyt and Sepaniak have used capillary zone electrophoresis to determine procaine in pharmaceuticals as a cation of benzylpenicillin [148]. A benzylpenicillin potassium tablet (250 mg) was treated with 20 mL of a 0.2% phenol solution (the internal standard), and dispersed in water. The solution was diluted to 500 mL, and samples were introduced into the fused silica capillary tube (70 cm x 50 gm) by siphoning. With 10 mM Na2HP04-6mM Na2B407 buffer as the mobile phase, the samples were subjected to electrophoresis at 30 kV (25 to 30 pA), and the emerging analytes detected at 228 nm within 10 minutes. 

Dose. E.V. and G.A. Guiochon Internal Standardization Techniques for Capillary Zone Electrophoresis. Anatvtical Chemistry. 1154 (June 1991). 

EV Koh, MG Bissell, RK Ito. Measurement of vitamin C by capillary electrophoresis in biological fluids and fruit beverages using a stereoisomer as an internal standard. J Chromatogr 633 245-250, 1993. 

Fig. 1. Purified proteins imaged using (A) Agilent 2100 Bioanalyzer and (B) traditional sodium dodecyl sulfide-polyacrylamide gel electrophoresis. (A) Note that the lowest two bands and the uppermost band are internal standards for sizing and quantitation. (B) 3-15% polyacrylamide gel stained with GelCode Blue. In both panels, the predicted size of the protein is labeled at the top of each lane.

Buffer Mixtures Commonly Used for Polyacrylamide Gel Electrophoresis Proteins for Internal Standardization of Polyacrylamide Gel Electrophoresis Chromogenic Stains for Gels Fluorescent Stains for Gels... 

The following table provides a list of proteins that may be used as internal standards, along with their isoelectric points, pi, in quantitative applications of polyacrylamide gel electrophoresis. These proteins may be used in isoelectric focusing or in SDS-PAGE. The isoelectric points are reported at 25°C.1... 

The analysis of aliphatic acids was performed using a P/ACE MDQ capillary electrophoresis instrument equipped with a 60 cm x 50 pm id fused silica capillary (Beckman Coulter, Fullerton, CA). The samples were filtered through a 0.45-gm cellulose acetate filter (Whatman, Maidstone, UK) prior to hydrodynamic injection at 15 psi for 4 s. The voltage was set to 20 kV at reversed polarity. The electrolyte, composed of 5.0 mM trimellitic acid, 50 mM tris(hydroxymethyl)-aminomethane, 1.0 mM tetradecyl-trimethylammoniumbromide, and 0.5 mM calcium chloride, had a pH of 9.8. Before use, it was filtered through a 0.2-gm cellulose nitrate filter and degassed withhelium. Detection was performedby indirect UV absorption at 220 nm. Succinic acid was used as internal standard. 

Sample preparation procedures for GC are generially more involved. For example, for methadone hydrochloride, 0.5jV sodium hydroxide is added to give the free base, followed by extraction with methylene chloride. An internal standard is added after the extract is dried with anhydrous sodium sulfate [3, p. 970]. The assay of interleukin-la formulated with human serum albumin does not require any sample treatment prior to analysis by capillary electrophoresis [44]. 

Capillary electrophoresis has been demonstrated to be useful in monitoring the identity and purity of hGH. CZE is capable of discrimination between hGH and several closely related impurities and degradation products, either as the intact species (previous work) or as the trypsin digests (this work). Thus, it is an important adjunct to conventional methods, such as RP-HPLC of digests or conventional electrophoresis of intact proteins, for the identification of hGH. CZE possesses adequate sensitivity to monitor minor impurities it is comparable to RP-HPLC with respect to linearity and precision. Samples with a volume of at least 10 nanoliters will provide acceptable precision those that contain an internal standard will provide the best precision. The peak area response is linear it is possible to extend linearity by increasing concentration as long as the contribution of the sample and its matrix to field inhomogeneities is minimized. The work shows that CZE has potential to be useful in the quality control of proteins such as hGH. 

At present, there are advanced difference gel electrophoresis (DOGE) Systems and 2-D fluorescence difference gel electrophoresis (2-D DIGE) which enable the analyst to use simultaneously modern (more precise) methods of fluorescent analysis with 2-D electrophoresis (using internal patterns), aided by a fully integrated bioinformatics system. Such systems allow more complete differential protein analysis, while the application of internal standards eliminates differentiation between the intervals, thus ensuring that even the smallest differences will be detected irrespective of the multitude of components. This guarantees reproducibility of results and their statistical reliability. Such assays are one of the platforms employed in the research based on the proteomics method. 

A mixture of four fluoroquinolones (including ciprofloxacin hydrochloride) was determined by high performance capillary electrophoresis using caffeine as an internal standard [58], A portion of the solution was... 

Ciprofloxacin and its metabolite desethylene-ciprofloxacin were determined in human plasma using capillary electrophoresis in the presence of N-(l-naphthyl) ethylenediamine dihydrochloride as an internal standard. Krol et al. [15] have thoroughly investigated the biotransformation of ciprofloxacin in body fluids using high performance liquid chromatography (HPLC) and proposed the sequence of metabolic pathways illustrated in Figure 1. 

Figure 3.2 Difference gel electrophoresis (DIGE). Ettan DIGE workflow three-color and two-color experiments including the internal standard. For fluorescence proteins tagging, two different CyDyes techniques are available. Minimal fluors allow consideration of three different CyDyes (Cy2, Cy3 and Cy5) in a multiplexing experiment.

Alban, A., David, S. O., Bjorkesten, L, Andeesson, C., Sloge, E., Lewis, S., Currie, I. (2003). A novel experimental design for comparative two-dimensional gel analysis two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36-44. 

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