Multiplexed Comparative Analysis of Intact Glycopeptides Using Electron-Transfer Dissociation and Synchronous Precursor Selection Based Triple-Stage Mass Spectrometry.

A recently developed synchronous precursor selection (SPS) mass spectrometry to the third (MS3) protocol enables more accurate multiplexed quantification of proteins/peptides using tandem mass tags (TMT) through comparison of reporter ion intensities at the MS3 level. However, challenges still exist for TMT-based simultaneous quantification and identification of intact glycopeptides due to inefficient peptide backbone fragmentation when using collision-induced dissociation (CID). To overcome this limitation, here we report an improved SPS/ETD workflow for TMT-based intact glycopeptide quantification and identification. The SPS/ETD approach was implemented on an Orbitrap Tribrid mass spectrometer and begins with selection of a parent ion in the MS scan, followed by tandem mass spectrometry (MS2) fragmentation by CID in the ion trap. Following MS2 fragmentation, SPS enables simultaneous isolation of the top 10 MS2 fragment ions for further higher energy collisional dissociation (HCD) fragmentation with the resulting MS3 fragments detected in an Orbitrap analyzer. Here, in addition to the standard SPS workflow, an electron-transfer dissociation (ETD) MS2 was performed and analyzed in the ion trap. The resultant ETD and CID spectra were used for the identification of the intact glycopeptides, while the quantitative comparison of site-specific glycans was achieved utilizing TMT reporter ions from HCD MS3 spectra. For intact glycopeptides, through systematic optimization and evaluation using a glycoprotein interference model, the SPS/ETD approach was demonstrated to offer improved accuracy, precision, and sensitivity compared to traditional data-dependent MS2 quantification, while maintaining the glycopeptide identification capability. Finally, this workflow was applied for the site-specific quantitative comparison of the glycoforms for two therapeutic enzymes (Cerezyme and VPRIV) and their different lots. The results demonstrate that this workflow is suitable for TMT-based intact glycopeptide characterization of glycoproteins.

Rapid screening and structural elucidation of a novel sibutramine analogue in a weight loss supplement: 11-desisobutyl-11-benzylsibutramine.

A novel analogue of sibutramine, 11-desisobutyl-11-benzylsibutramine, has been discovered. During routine ion mobility spectrometry (IMS) screening of a weight loss supplement collected at an US FDA import operation facility an unknown peak was observed. Further analysis of the supplement by liquid chromatography-mass spectrometry (LC-MS) and high resolution mass spectrometry revealed an unknown peak with a relative retention time of 1.04 with respect to sibutramine and a predicted formula of C20H24NCl. In order to elucidate the analogue's structure, it was isolated from the supplement and characterized by tandem mass spectrometry and nuclear magnetic resonance (NMR), which revealed the analogue possessed a benzyl moiety at the 11 position in place of the isobutyl group associated with sibutramine.

Rapid-screening detection of acetildenafils, sildenafils and avanafil by ion mobility spectrometry.

Ion mobility spectrometry was used as a rapid screening tool for the detection of acetildenafils, sildenafils and avanafil within adulterated herbal supplement matrices. Acetildenafils show a tendency for partial fragmentation during the desorption/ionization process affording two peaks in the ion mobility spectrum in addition to the intact compound. The fragmentation appears to occur α to the carbonyl group along the CN bond attaching the piperazine moiety, producing a common fragment (K0=1.0280 cm²V⁻¹s⁻¹) along with the respective piperazine fragment. The sildenafils and avanafil afford one molecular ion peak per compound.

Qualitative screening for adulterants in weight-loss supplements by ion mobility spectrometry.

Ion mobility spectrometry (IMS) served as a rapid, qualitative screening tool for the analysis of adulterated weight-loss products. We have previously shown that sibutramine extracted into methanol from dietary supplements can be detected at low levels (2ng) using a portable IMS spectrometer, and have adapted a similar method for the analysis of additional weight-loss product adulterants. An FDA collaborative study helped to define the limits for fluoxetine with a limit of detection of 2ng. We also evaluated more readily available, less toxic extraction solvents and found isopropanol and water were comparable to methanol. Isopropanol was favored over water for two reasons: (1) water increases the analysis time and (2) aqueous solutions were more susceptible to pH change, which affected the detection of sibutramine. In addition to sibutamine and fluoxetine, we surveyed 11 weight-loss adulterants; bumetanide, fenfluramine, furosemide, orlistat, phenolphthalein, phentermine, phenytoin, rimonabant, sertraline and two sibutramine analogs, desmethylsibutramine and didesmethylsibutramine, using portable and benchtop ion mobility spectrometers. Out of these 13 active pharmaceutical ingredients (APIs), portable and benchtop ion mobility spectrometers were capable of screening products for 10 of these APIs. The developed procedure was applied to two weight-loss dietary supplements using both portable and benchtop instruments. One product contained didesmethylsibutramine while the other contained didesmethylsibutramine and phenolphthalein.

Selective melamine detection in multiple sample matrices with a portable Raman instrument using surface enhanced Raman spectroscopy-active gold nanoparticles.

Melamine adulteration of food and pharmaceutical products is a major concern and there is a growing need to protect the public from exposure to contaminated or adulterated products. One approach to reduce this threat is to develop a portable method for on-site rapid testing. We describe a universal and selective method for the detection of melamine in a variety of solid matrices at the 100-200 µg L(-1) level by surface enhanced Raman spectroscopy (SERS) with gold nanoparticles. With minimal sample preparation and the use of a portable Raman spectrometer, this work will lead to field-based screening for melamine adulteration. Citrate coated gold nanoparticles (Au NPs) were investigated for both colorimetric and Raman-based responses. Several non-hazardous solvents were evaluated in order to develop a melamine extraction procedure safe for field applications. Au NP agglomerates formed by the addition of isopropanol (IPA) prior to sample introduction enhanced the Raman signal for melamine and eliminated matrix interference for substrate formation. The melamine Raman signal resulted in a 10(5) enhancement through the use of Au NP agglomerates. To our knowledge, we have developed the first portable SERS method using Au NPs to selectively screen for the presence of melamine adulteration in a variety of food and pharmaceutical matrices, including milk powder, infant formula, lactose, povidone, whey protein, wheat bran and wheat gluten.

Libraries, classifiers, and quantifiers: a comparison of chemometric methods for the analysis of Raman spectra of contaminated pharmaceutical materials.

In this study, pharmaceutical grade sorbitol was used as a model system for comparison of Raman based library spectral correlation methods with more sophisticated methods of chemometric data analysis. Both crystallizing sorbitol (CS) and non-crystallizing sorbitol (NCS) from several manufacturers were examined. The Raman spectrum of each sample was collected and identified by correlation with a spectral library that included the CS spectrum but not the NCS spectrum. The average hit quality index (HQI) for the measured NCS spectra and the library CS spectrum was 0.966 whereas the average HQI for the measured CS spectra was 0.991. Both HQIs exceeded the 0.950 threshold that is commonly used for material verification. To enhance the discrimination between CS and NCS, a CS/NCS classification model was constructed using soft independent modeling of class analogies (SIMCA). SIMCA was able to positively identify CS and NCS solutions with no misclassifications. When CS was adulterated with low levels (0-5%) of ethylene glycol (EG) and diethylene glycol (DEG), the HQI values of the measured spectra and the CS library spectrum were still above 0.950. When the CS SIMCA model was applied to adulterated CS spectra, it determined that CS samples with adulterant levels as low as 2% were outside of the CS class. A quantitative PLS model was also applied to EG adulterated CS and resulted in a detection limit of 0.9% for EG. The results obtained from these studies highlight the importance of selecting an appropriate data analysis process for the detection of low level adulterants in pharmaceutical raw materials using Raman spectroscopic screening methods.

Multivariate calibration and instrument standardization for the rapid detection of diethylene glycol in glycerin by Raman spectroscopy.

The transfer of a multivariate calibration model for quantitative determination of diethylene glycol (DEG) contaminant in pharmaceutical-grade glycerin between five portable Raman spectrometers was accomplished using piecewise direct standardization (PDS). The calibration set was developed using a multi-range ternary mixture design with successively reduced impurity concentration ranges. It was found that optimal selection of calibration transfer standards using the Kennard-Stone algorithm also required application of the algorithm to multiple successively reduced impurity concentration ranges. Partial least squares (PLS) calibration models were developed using the calibration set measured independently on each of the five spectrometers. The performance of the models was evaluated based on the root mean square error of prediction (RMSEP), calculated using independent validation samples. An F-test showed that no statistical differences in the variances were observed between models developed on different instruments. Direct cross-instrument prediction without standardization was performed between a single primary instrument and each of the four secondary instruments to evaluate the robustness of the primary instrument calibration model. Significant increases in the RMSEP values for the secondary instruments were observed due to instrument variability. Application of piecewise direct standardization using the optimal calibration transfer subset resulted in the lowest values of RMSEP for the secondary instruments. Using the optimal calibration transfer subset, an optimized calibration model was developed using a subset of the original calibration set, resulting in a DEG detection limit of 0.32% across all five instruments.

Using a portable ion mobility spectrometer to screen dietary supplements for sibutramine.

In response to recent incidents of undeclared sibutramine, an appetite suppressant found in dietary supplements, we developed a method to detect sibutramine using hand-held ion mobility spectrometers with an analysis time of 15 s. Ion mobility spectrometry is a high-throughput and sensitive technique that has been used for illicit drug, explosive, volatile organic compound and chemical warfare detection. We evaluated a hand-held ion mobility spectrometer as a tool for the analysis of supplement extracts containing sibutramine. The overall instrumental limit of detection of five portable ion mobility spectrometers was 2 ng of sibutramine HCl. When sample extractions containing 30 ng/µl or greater of sibutramine were analyzed, saturation of the ionization chamber of the spectrometer occurred and the instrument required more than three cleaning cycles to remove the drug. Hence, supplement samples suspected of containing sibutramine should be prepared at concentrations of 2-20 ng/µl. To obtain this target concentration range for products containing unknown amounts of sibutramine, we provided a simple sample preparation procedure, allowing the U.S. Food and Drug Administration or other agencies to screen products using the portable ion mobility spectrometer.