Changes in composition and sulfation patterns of glycoaminoglycans in renal cell carcinoma.

Glycosaminoglycans (GAGs) are heterogeneous, linear, highly charged, anionic polysaccharides consisting of repeating disaccharides units. GAGs have some biological significance in cancer progression (invasion and metastasis) and cell signaling. In different cancer types, GAGs undergo specific structural changes. In the present study, in depth investigation of changes in sulfation pattern and composition of GAGs, heparan sulfate (HS)/heparin (HP), chondroitin sulfate (CS)/dermatan sulfate and hyaluronan (HA) in normal renal tissue (NRT) and renal cell carcinoma tissue (RCCT) were evaluated. The statistical evaluation showed that alteration of the HS (HSNRT = 415.1 ± 115.3; HSRCCT = 277.5 ± 134.3), and CS (CSNRT = 35.3 ± 12.3; CSRCCT = 166.7 ± 108.8) amounts (in ng/mg dry tissue) were statistically significant (p < 0.05). Sulfation pattern in NRT and RCCT was evaluated to reveal disaccharide profiles. Statistical analyses showed that RCCT samples contain significantly increased amounts (in units of ng/mg dry tissue) of 4SCS (NRT = 25.7 ± 9.4; RCCT = 117.1 ± 73.9), SECS (NRT = 0.7 ± 0.3; RCCT = 4.7 ± 4.5), 6SCS (NRT = 6.1 ± 2.7; RCCT = 39.4 ± 34.7) and significantly decreased amounts (in units of ng/mg dry tissue) of NS6SHS (RCCT = 28.6 ± 6.5, RCCT = 10.2 ± 8.0), NS2SHS (RCCT = 44.2 ± 13.8; RCCT = 27.2 ± 15.0), NSHS (NRT = 68.4 ± 15.8; RCCT = 50.4 ± 21.2), 2S6SHS (NRT = 1.0 ± 0.4; RCCT = 0.4 ± 0.3), and 6SHS (NRT = 60.6 ± 17.5; RCCT = 24.9 ± 12.3). If these changes in GAGs are proven to be specific and sensitive, they may serve as potential biomarkers in RCC. Our findings are likely to help us to show the direction for further investigations to be able to bring different diagnostic and prognostic approaches in renal tumors.

Capillary electrophoresis for total glycosaminoglycan analysis.

A capillary zone electrophoresis-laser-induced fluorescence detection (CZE-LIF) method was developed for the simultaneous analysis of disaccharides derived from heparan sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan, and keratan sulfate. Glycosaminoglycans (GAGs) were first depolymerized with the mixture of GAG lyases (heparinase I, II, III and chondroitinase ABC and chondroitinase AC II) and GAG endohydrolase (keratinase II) and the resulting disaccharides were derivatized by reductive amination with 2-aminoacridone. Nineteen fluorescently labeled disaccharides were separated using 50 mM phosphate buffer (pH 3.3) under reversed polarity at 25 kV. Using these conditions, all the disaccharides examined were baseline separated in less then 25 min. This CZE-LIF method gave good reproducibility for both migration time (≤1.03% for intraday and ≤4.4% for interday) and the peak area values (≤5.6% for intra- and ≤8.69% for interday). This CZE-LIF method was used for profiling and quantification of GAG derivative disaccharides in bovine cornea. The results show that the current CZE-LIF method offers fast, simple, sensitive, reproducible determination of disaccharides derived from total GAGs in a single run.

Ultrasensitive detection and quantification of acidic disaccharides using capillary electrophoresis and quantum dot-based fluorescence resonance energy transfer.

Rapid and highly sensitive detection of the carbohydrate components of glycoconjugates is critical for advancing glycobiology. Fluorescence (or Förster) resonance energy transfer (FRET) is commonly used in detection of DNA, in protein structural biology, and in protease assays but is less frequently applied to glycan analysis due to difficulties in inserting two fluorescent tags into small glycan structures. We report an ultrasensitive method for the detection and quantification of a chondroitin sulfate disaccharide based on FRET, involving a CdSe-ZnS core-shell nanocrystal quantum dot (QD) streptavidin conjugate donor and a Cy5 acceptor. The disaccharide was doubly labeled with biotin and Cy5. QDs then served to concentrate the target disaccharide, enhancing the overall energy transfer efficiency, with unlinked QDs and Cy5 hydrazide producing nearly zero background signal in capillary electrophoresis using laser-induced fluorescence detection with two different band-pass filters. This method is generally applicable to the ultrasensitive analysis of acidic glycans and offers promise for the high-throughput disaccharide analysis of glycosaminoglycans.

Analysis of glycoforms on the glycosylation site and the glycans in monoclonal antibody biopharmaceuticals.

Therapeutic monoclonal antibodies (mAbs), immunoglobulins, have been efficiently used in the treatment of many diseases, such as cancer, inflammatory and cardiovascular diseases, and organ transplantation. mAbs are glycoprotein molecules undergoing posttranslational modifications. Glycosylation is one of the posttranslational modifications. Different glycoforms that are important for maintaining the potency of mAb drugs show various biological activities. Therefore, the profile of the glycans and glycosylation sites should be determined to produce safe, good quality, consistent mAb drugs for human use. For this reason, simple, robust, accurate, and reproducible analytical methods need to be developed. In this article, chromatographic methods for the analysis of the glycoforms on the glycosylation site and the glycans in mAb biopharmaceuticals have been evaluated.

Quantitative analysis of ezetimibe in human plasma by gas chromatography-mass spectrometry.

A new, specific and sensitive GC-MS method with electron impact ionization technique was developed for quantitative analysis of ezetimibe (EZE) in human plasma. Prior to GC analysis, EZE was derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA), which is a trimethyl silylating reagent. The derivatization reaction was optimized and parameters such as catalyst, derivatization time, temperature, solvent and the volume of silylating reagent were investigated. Trimethylsilyl ether derivative of EZE was determined in selected ion monitoring (SIM, mass-to-charge ratio (m/z): 326) mode. The method was validated with respect to LOD and LOQ, precision, accuracy, linearity, specificity, stability, and recovery. The LOQ and LOD were found as 15 and 10 ng/mL, respectively. The linearity of the method ranged from 15 to 250 ng/mL. The correlation coefficient of the calibration curve was 0.9977 +/- 0.0004 (+/- S.E.M.). The intra- and inter-day precisions (RSD) were less than 6% and accuracies (bias) for intra- and inter-day accuracy were found between -4.04 and 9.71% at four different concentration levels (15, 40, 100, 250 ng/mL). The proposed method was successfully applied to real human plasma samples for determination of total EZE.

Development of Sensitive and Specific Analysis of Vildagliptin in Pharmaceutical Formulation by Gas Chromatography-Mass Spectrometry.

A sensitive and selective gas chromatography-mass spectrometry (GC-MS) method was developed and fully validated for the determination of vildagliptin (VIL) in pharmaceutical formulation. Prior to GC-MS analysis, VIL was efficiently derivatized with MSTFA/NH4I/ß-mercaptoethanol at 60°C for 30 min. The obtained O-TMS derivative of VIL was detected by selected ion monitoring mode using the diagnostic ions m/z 223 and 252. Nandrolone was chosen as internal standard. The GC-MS method was fully validated by the following validation parameters: limit of detection (LOD) and quantitation (LOQ), linearity, precision, accuracy, specificity, stability, robustness, and ruggedness. LOD and LOQ were found to be 1.5 and 3.5 ng mL(-1), respectively. The GC-MS method is linear in the range of 3.5-300 ng mL(-1). The intra- and interday precision values were less than ≤3.62%. The intra- and interday accuracy values were found in the range of -0.26-2.06%. Finally, the GC-MS method was successfully applied to determine VIL in pharmaceutical formulation.

Development of a gas chromatography-mass spectrometry method for the analysis of sitagliptin in human urine.

A new, simple and rapid gas chromatography-mass spectrometry (GC-MS) method for the determination of sitagliptin (STG) in human urine was developed and fully validated. STG was derivatized by N-methyl-trimethylsilyltrifluoroacetamide prior to GC-MS analysis and converted to its N-TMS amine derivative. It was extracted from urine by using carbonate buffer (pH 9.0) and ether. The method was validated in terms of specificity, limit of quantitation (LOQ), linearity, accuracy, precision, stability, recovery, robustness and ruggedness. LOQ was found to be 50 ng mL(-1). The calibration curve was linear in the range of 50-600 ng mL(-1) with a coefficient of determination (r(2)) above 0.997. The intra- and inter-day precisions were less than 8.76%, and the intra- and inter-day accuracies were found between 0.83 and 4.53%. The method was successfully applied to urine samples obtained from diabetic patients.