LC Chromatogram Research Articles

Simultaneous analysis of cannabinoids and terpenes in Cannabis sativa inflorescence using full comprehensive two-dimensional liquid chromatography coupled to smart active modulation

Nowadays, the higher peak capacity achievable by comprehensive two-dimensional liquid chromatography (LC×LC) for the analysis of vegetal samples is well-recognized. In addition, numerous compounds may be present in very different amounts. Cannabinoids and terpenes represent the main components of Cannabis sativa inflorescence samples, whose quantities are relevant for many application purposes. The analyses of both families are performed by different methods, at least two different separation methodologies, mainly according to their chemical characteristics and concentration levels. In this work, concentration differences and sample complexity issues were addressed using an LC×LC method that incorporates an optimized modulation strategy, namely smart active modulation, for the simultaneous analysis of cannabinoids and terpenes. The system was built by interposing an active flow splitter pump between both dimensions. This set up aimed to exploit the known advantages of LC×LC. In addition, here we proposed to use the splitter pump for online control over the splitting ratio to facilitate the selective dilution of different eluted fractions containing compounds with highly different concentrations.This work represents the first application and demonstration of smart active modulation (SAM) in LC×LC to simultaneously determine analytes with significant differences in concentration levels present in complex samples. The proposed method was tested with eight different strains, from which fingerprints were taken, and numerous cannabinoids and terpenes were identified in these samples. With this strategy, between 49 and 54 peaks were obtained in the LC×LC chromatograms corresponding to different strains. THCA-A was the main component in six strains, while CBDA was the main component in the other two strains. The main terpenes found were myrcene (in five strains), limonene (in two strains), and humulene (in one strain). Additionally, numerous other cannabinoids and terpenes were identified in these samples, providing valuable compositional information for growers, as well as medical and recreational users. The SAM strategy here proposed is simple and it can be extended to other complex matrices.

Sensitive and Rapid LCMS/MS Method for the Estimation of recently approved Antiviral drugs Maribavir and Fostemsavir in spiked human plasma

In this study, a straightforward, highly sensitive, and selective liquid chromatography/tandem mass spectrometry (LC–MS/MS) method was developed and rigorously validated for the simultaneous quantification of Maribavir and Fostemsavir in human plasma. To ensure precision and reliability, we employed Dolutegravir as the internal standard (IS). The analytical process involved a two-step extraction method. Initially, protein precipitation was induced by the addition of acetonitrile, followed by liquid–liquid extraction using a 1:1 (v/v) mixture of diethyl ether and dichloromethane as the extracting solvent. Separation of the analytes was achieved through reversed phase high-performance liquid chromatography (HPLC) using a Phenomenex C18 Luna column (4.6 mm×100 mm, 5 µm). A simple isocratic mobile phase consisting of acetonitrile, methanol, and 0.1% formic acid (35:55:10, v/v) was used, operating at a flow rate of 0.5 mL/min. Under these optimized conditions, the LC chromatogram of the spiked standard exhibited distinct peaks at retention times of 2.07 min, 2.59 min, and 4.29 min for Fostemsavir, Maribavir, and the internal standard, respectively. Detection was performed using a triple quadrupole mass spectrometer employing electrospray ionization in positive ion mode and multiple reaction monitoring (MRM) mode. The mass transitions monitored were m/z 377 → 110, m/z 584 → 105, and m/z 420 → 142 for maribavir, fostemsavir, and dolutegravir, respectively. This method provided a rapid analysis within 5 minutes, over a linear concentration range of 15-750 ng/mL for both maribavir and fostemsavir. Method validation was conducted following FDA guidelines for bio-analytical methods, and the results consistently fell within the acceptable limits for both analytes. Therefore, our developed method holds promise for the accurate analysis of maribavir and fostemsavir in human plasma, and it has potential applications in pharmacokinetic studies

Biotransformation of Barley Phenolamide by Mice and the Human Gut Microbiota and Quantitative Analysis of the Major Metabolites in Mice

This study investigates the metabolism of p-coumaroylagmatine (pCAA), one of the phenolamides in barley, in mice, and by human gut microbiota, and measures the concentrations of its main metabolites in mice. Nine major metabolites are identified from fecal and urinary samples collected from pCAA treated mice via analysis of their LC chromatograms and tandem mass spectra compared to the commercial and synthesized standards. These nine metabolites are generated through four different biotransformation pathways: double bond reduction, amide bond hydrolyzation, cleavage of guanidine, and oxidation of guanidine. Furthermore, interindividual differences in the formation of dihydro-pCAA (M3), high and low metabolizers, are observed in human in vitro intestinal microbial conversion. Moreover, significant amount of pCAA is detected in mice (29.33± 1.58µmolg-1 in feces and 2020.44± 130.07µM in urine), and the concentrations of agmatine (M1) are increased to 177.6 times and 3.2 times in mouse feces and urine, respectively. This study demonstrates that pCAA is metabolized in mice and by human gut microbiota to generate potential bioactive metabolites through four major metabolic pathways. pCAA and its metabolites have the potential to be used as the exposure biomarkers to reflect the intake of whole grain barley.

VerA Gene is Involved in the Step to Make the Xanthone Structure of Demethylsterigmatocystin in Aflatoxin Biosynthesis.

In the biosynthesis of aflatoxin, verA, ver-1, ordB, and hypA genes of the aflatoxin gene cluster are involved in the pathway from versicolorin A (VA) to demethylsterigmatocystin (DMST). We herein isolated each disruptant of these four genes to determine their functions in more detail. Disruptants of ver-1, ordB, and hypA genes commonly accumulated VA in their mycelia. In contrast, the verA gene disruptant accumulated a novel yellow fluorescent substance (which we named HAMA) in the mycelia as well as culture medium. Feeding HAMA to the other disruptants commonly caused the production of aflatoxins B1 (AFB1) and G1 (AFG1). These results indicate that HAMA pigment is a novel aflatoxin precursor which is involved at a certain step after those of ver-1, ordB, and hypA genes between VA and DMST. HAMA was found to be an unstable substance to easily convert to DMST and sterigmatin. A liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular mass of HAMA was 374, and HAMA gave two close major peaks in the LC chromatogram in some LC conditions. We suggest that these peaks correspond to the two conformers of HAMA; one of them would be selectively bound on the substrate binding site of VerA enzyme and then converted to DMST. VerA enzyme may work as a key enzyme in the creation of the xanthone structure of DMST from HAMA.

Separation and identification of the constituents in Fangchi Radix of different origins

Fangchi Radix is derived from Aristolochiaceae or Menispermaceae plants and contains a series of biologically active alkaloids. Fifteen alkaloids in Fangchi crude extracts, including acutumidine(1), magnoflorine(2), stepharine(3), sinomenine(4), acutumine(5), cyclanoline(6), fangchinoline(7), berbamine(8), tetrandrine(9), isotetrandrine(10), trilobine(11), aristolochic acid II(12), aristolochic acid I(13), aristololactam(14) and isotrilobine(15), were separated completely by high-performance liquid chromatography (HPLC) within 60 min. UV detection at 254 nm with a linear gradient elution consisting of 20 mM CH3COONH4 (pH 4.81) and H2O-CH3CN was found to be the most efficient eluent for the LC and LC-MS separations. The structures of the compounds corresponding to the peaks in the LC chromatogram were identified by LC-MS. A total of 32 commercial samples of Fangchi Radix from Sinomenium acutumn, Stephania tetrandra, Aristolochia fangchi and Cocculus trilobus were collected from herbal markets in Taiwan, Japan and China. The contents (peak-area ratios) and structures of the fifteen constituents in these samples were readily determined by the proposed methods and the results could be used to estimate the origin and quality of an herbal drug.

Determination of Quercetin and Flavonol Synthase in Boesenbergia rotunda Rhizome.

Flavonols in plants are catalyzed by flavonol synthase (FLS) enzyme. FLS was reported expressed in flowers and fruits, i.e., Dianthus caryophyllus L. (Caryophyllaceae), Petunia hybrida Hort. (Solanaceae), Arabidopsis thaliana L. (Brassicaceae), Citrus unshiu Marc. (Rutaceae). However, none reported about FLS in medicinal plants, particularly those which possess anti-inflammatory activity. This study was aimed to extract and identify FLS in the rhizome of Boesenbergia rotunda (Zingiberaceae) and to determine quercetin in the ethanol extract of the rhizome. The protein extraction of the rhizome was carried out by employing Laing and Christeller's (2004) and Wang's (2014) methods. The extracted-proteins were separated by using SDS-PAGE, followed by the measurement of FLS intensity by using Gel Analyzer. The FLS-1 of recombinant A. thaliana was employed as the standard. The determination of quercetin in the rhizome was carried out using LC-MS. The FLS occurred as a thick band at 38 kDa with intensity 116-158. The LC chromatogram of the extract indicated a small peak at 7.94 min similar to that of quercetin standard. The MS spectra at 7.94 min indicated that quercetin is present in the B. rotunda rhizome (m/z = 303.0549). The concentration of quercetin in the extract is 0.022% w/v. The FLS, an enzyme which plays an important role in producing quercetin, was detected in B. rotunda rhizome planted in Indonesia. As a consequence, quercetin in a small amount, was also quantified in the rhizome of this plant. This report will add a scientific insight of B. rotunda for biological sciences.

A UHPLC-UV Method Development and Validation for Determining Kavalactones and Flavokavains in Piper methysticum (Kava).

An ultra-high-performance liquid chromatographic (UHPLC) separation was developed for six kava pyrones (methysticin, dihydromethysticin (DHM), kavain, dihydrokavain (DHK), desmethoxyyangonin (DMY), and yangonin), two unidentified components, and three Flavokavains (Flavokavain A, B, and C) in Piper methysticum (kava). The six major kavalactones and three flavokavains are completely separated (Rs > 1.5) within 15 min using a HSS T3 column and a mobile phase at 60 °C. All the peaks in the LC chromatogram of kava extract or standard solutions were structurally confirmed by LC-UV-MS/MS. The degradations of yangonin and flavokavains were observed among the method development. The degradation products were identified as cis-isomerization by MS/MS spectra. The isomerization was prevented or limited by sample preparation in a non-alcoholic solvent or with no water. The method uses the six kava pyrones and three flavokavains as external standards. The quantitative calibration curves are linear, covering a range of 0.5–75 μg/mL for the six kava pyrones and 0.05–7.5 μg/mL for the three flavokavains. The quantitation limits for methysticin, DHM, kavain, DHK, DMY, and yangonin are approximately 0.454, 0.480, 0.277, 0.686, 0.189, and 0.422 μg/mL. The limit of quantification (LOQs) of the three flavokavains are about 0.270, 0.062, and 0.303 μg/mL for flavokavain C (FKC), flavokavain A (FKA), and flavokavain B (FKB). The average recoveries at three different levels are 99.0–102.3% for kavalactones (KLs) and 98.1–102.9% for flavokavains (FKs). This study demonstrates that the method of analysis offers convenience and adequate sensitivity for determining methysticin, DHM, kavain, DHK, yangonin, DMY, FKA, FKB, and FKC in kava raw materials (root and CO2 extract) and finished products (dry-filled capsule and tablet).

Online Separation and Identification of Isomers Using Infrared Multiple Photon Dissociation Ion Spectroscopy Coupled to Liquid Chromatography: Application to the Analysis of Disaccharides Regio-Isomers and Monosaccharide Anomers.

The vast array of molecular isomerisms which form the complex molecular structure of carbohydrates is the foundation of their biological versatility but defies the analytical chemist. Hyphenations of mass spectrometry with orthogonal structural characterization, such as ion mobility or ion spectroscopy, have recently shown great promise for distinction between closely related molecular structures. Yet, the lack of analytical strategies for identification of isomers present in mixtures remains a major obstacle to routine carbohydrate sequencing. In this context, an ideal workflow for glycomics would combine isomer separation and individual characterization of the molecular structure with atomistic resolution. Here we report the implementation of such a multidimensional analytical strategy, which consists of the first online coupling of high-performance liquid chromatography (HPLC)-MS and infrared multiple photon dissociation (IRMPD) spectroscopy. The performance of this novel workflow is exemplified in the case of monosaccharides (anomers) and disaccharides (regioisomers) standards. We report that the LC-MS-IRMPD approach offers a robust advanced MS diagnostic of mixtures of isomers, including carbohydrate anomers, which is critical for carbohydrate sequencing. Our results also explain the bimodal character generally observed in LC chromatograms of carbohydrates. More generally, this multidimensional analytical strategy opens the gateway to rapid identification of molecular isoforms with potential application in the "omics" fields.

Development of a novel HPLC method for the determination of the impurities in desonide cream and characterization of its impurities by 2D LC-IT-TOF MS

A novel HPLC method for the determination of the impurities in desonide cream was established and validated for the further improvement of the official monograph in USP. Desonide was well resolved from the photodegradation impurity, which overlapped with desonide in USP method. The method was validated in accordance to the regulatory guidelines recommended by the International Conference on Harmonisation and this validation included specificity, limit of detection, limit of quantification, linearity and accuracy. Four degradation impurities in desonide cream were characterized by a trap-free two-dimensional liquid chromatography coupled to high resolution ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) in positive mode of electrospray ionization. Through the multiple heart-cutting 2D LC approach and online demineralization technique, the problem of incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely, and the TIC chromatogram of LC–MS could be in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities. In the first dimension, the column was Phenomenex Kinetex C8 (4.6 mm × 150 mm, 2.6 μm) with a non-volatile salt mobile phase. In the second dimension, the column was Shimadzu Shim-pack GISS C18 (50 mm × 2.1 mm, 1.9 μm) with a volatile salt mobile phase. The structures of four degradation impurities in desonide cream were deduced based on the HPLC-MSn data. The established method in this study was simple and reliable for routine quality control of desonide cream.

Establishment of Liquid Chromatography Retention Index Based on Chemical Labeling for Metabolomic Analysis.

Chemical labeling (CL) in combination with liquid chromatography-mass spectrometry (LC-MS) analysis has been demonstrated to be a promising technology in metabolomic analysis. However, identification of chemically labeled metabolites remains to be challenging. Retention time (RT) is one of the most important parameters for the identification of metabolites, but it could vary greatly in LC-MS analysis. In this work, we developed a chemical labeling-based HPLC retention index (CL-HPLC RI) strategy to facilitate the identification of metabolites. In this CL-HPLC RI strategy, a series of 2-dimethylaminoethylamine (DMED)-labeled fatty acids were used as calibrants to establish RIs for DMED-labeled carboxylated compounds and a series of 4-( N, N-dimethylamino)phenyl isothiocyanate (DMAP)-labeled fatty amines were used as calibrants for DMAP-labeled amine compunds. To calculate the RIs, the whole LC chromatogram was divided into 24 time intervals by 23 DMED-labeled fatty acid standards or 15 time intervals by 14 DMAP-labeled fatty amine standards. Then, we established the RIs of 854 detected DMED-labeled carboxylated metabolites and 1057 DMAP-labeled amine metabolites in fecal samples and demonstrated that RIs were highly reproducible under different elution gradients, columns, and instrument systems. Finally, we applied this strategy to the identification of metabolites in human serum. Using RIs, 267 DMED-labeled carboxylated metabolites and 273 DMAP-labeled amine metabolites in human serum matched well with the fecal metabolome database. Taken together, the developed CL-HPLC RI strategy was demonstrated to be a promising method to facilitate the identification of metabolites in metabolomic analysis.

Application of a trap-free two-dimensional liquid chromatography combined with ion trap/time-of-flight mass spectrometry for separation and characterization of impurities and isomers in cefpiramide

High-resolution mass spectrometry had been routinely used for structure identification of impurity. However, all LC-MS methods were based on a volatile mobile phase, and a non-volatile system is used in the official analytical method of United States Pharmacopoeia for cefpiramide which limited the use of mass spectrometry for structure characterization of the impurities. Here we presented the utilization of a trap-free two-dimensional liquid chromatography coupled to high resolution ion trap/time-of-flight mass spectrometry (2D LC-IT-TOF MS) with positive and negative modes of electrospray ionization for characterization of eight impurities in cefpiramide. Trap-free two-dimensional liquid chromatography and online desalting technique made it possible to characterize the impurity in cefpiramide in the condition of official standard, and the TIC chromatogram of LC-MS was in conformity with the LC chromatogram of the official analytical method in the peak sequence of impurities, which could further improve the method of official monographs in pharmacopoeias. Each peak separated by the non-volatile mobile phase was trapped by a 20 μL quantitative loop then transferred into a system with a volatile mobile phase connected to a MS detector. In the first dimension, the column was Kromasil C8 analytical column (250 mm × 4.6 mm, 5 μm) with a non-volatile salt mobile phase at the flow rate of 0.8 mL min−1. In the second dimension, the column was Shimadzu Shim-pack GISS C18 (50 mm × 2.1 mm, 1.9 μm) with a volatile salt mobile phase at the flow rate of 0.3 mL min−1. Through the multiple heart-cutting 2D-LC approach and online desalting technique, the problem of incompatibility between non-volatile salt mobile phase and mass spectrometry was solved completely. The fragmentation behavior of cefpiramide and its eight impurities were studied. The structures of eight impurities in cefpiramide drug substance were deduced based on the HPLC-MSn data, in which seven impurities were novel impurities. The forming mechanisms of degradation products in cefpiramide were also studied.

Separation and characterization of unknown impurities in cefonicid sodium by trap-free two-dimensional liquid chromatography combined with ion trap time-of-flight mass spectrometry.

Seven unknown antibiotic impurities in cefonicid sodium were separated and characterized by a trap-free two-dimensional liquid chromatography coupled to high-resolution ion-trap time-of-flight mass spectrometry (2D-LC/IT-TOF MS) using both positive and negative modes of electrospray ionization. Trap-free 2D-LC and an online demineralization technique made it possible to characterize cefonicid sodium under the conditions of the official standard, and the TIC chromatogram obtained by LC/MS was in conformity with the LC chromatogram obtained by the official analytical method in the peak sequence of impurities. In the first dimension, the column was a GRACE Alltima C18 (250mm×4.6mm, 5μm), and the gradient elution used 0.02mol·L-1 ammonium dihydrogen phosphate solution and methanol as mobile phase. In the second dimension, the analytical column was a Shimadzu Shim-pack GISS C18 (50mm×2.1mm, 1.9μm) with 10mmol·L-1 ammonium formate solution and methanol as mobile phase. Full scan LC/MS was first executed to obtain the exact m/z values of the molecules. Then LC/MS2 and LC-MS3 experiments were performed on the compounds of interest. The structures of seven unknown degradation products in cefonicid sodium were deduced based on the high-resolution MSn data using both positive and negative mode. The problem of incompatibility between the non-volatile salt mobile phase and mass spectrometry was solved completely by multidimensional heart-cutting approaches and an online demineralization technique, which is worthy of widespread use and application for the advantages of stability and repeatability.

Development of a hydrophilic interaction liquid chromatography coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging platform for N-glycan relative quantitation using stable-isotope labeled hydrazide reagents.

In this work, the capability of newly developed hydrophilic interaction liquid chromatography (HILIC) coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) platform for quantitative analysis of N-glycans has been demonstrated. As a proof-of-principle experiment, heavy and light stable-isotope labeled hydrazide reagents labeled maltodextrin ladder were used to demonstrate the feasibility of the HILIC-MALDI-MSI platform for reliable quantitative analysis of N-glycans. MALDI-MSI analysis by an Orbitrap mass spectrometer enabled high-resolution and high-sensitivity detection of N-glycans eluted from HILIC column, allowing the re-construction of LC chromatograms as well as accurate mass measurements for structural inference. MALDI-MSI analysis of the collected LC traces showed that the chromatographic resolution was preserved. The N-glycans released from human serum was used to demonstrate the utility of this novel platform in quantitative analysis of N-glycans from a complex sample. Benefiting from the minimized ion suppression provided by HILIC separation, comparison between MALDI-MS and the newly developed platform HILIC-MALDI-MSI revealed that HILIC-MALDI-MSI provided higher N-glycan coverage as well as better quantitation accuracy in the quantitative analysis of N-glycans released from human serum. Graphical abstract Reconstructed chromatograms based on HILIC-MALDI-MSI results of heavy and light labeled maltodextrin enabling quantitative glycan analysis.

Comparison of multivariate curve resolution strategies in quantitative LCxLC: Application to the quantification of furanocoumarins in apiaceous vegetables

Comprehensive two-dimensional liquid chromatography (LC × LC) has been gaining popularity for the analysis of complex samples in a wide range of fields including metabolomics, environmental analysis, and food analysis. While LC × LC can provide greater chromatographic resolution than one-dimensional LC (1D-LC), overlapping peaks are often still present in separations of complex samples, a problem that can be alleviated by chemometric curve resolution techniques such as multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS has also been previously shown to assist in the quantitative analysis of LC x LC data by isolating pure analyte signals from background signals which are often present at higher levels in LC x LC compared to 1D-LC. In this work we present the analysis of a dataset from the LC × LC analyses of parsley, parsnip and celery samples for the presence and concentrations of 14 furanocoumarins. Several MCR-ALS implementations are compared for the analysis of LC × LC data. These implementations include analyzing the LC x LC chromatogram alone, analyzing the one-dimensional chromatogram alone, as well as two hybrid approaches that make use of both the first and second dimension chromatograms. Furthermore, we compared manual integration of resolved chromatograms versus a simple summation approach, using the resolved chromatographic peaks in both cases. It is found that manual integration of the resolved LC × LC chromatograms provides the best quantification as measured by the consistency between replicate injections. If the summation approach is desired for automation, the choice of MCR-ALS implementation has a large effect on the precision of the analysis. Based on these results, the concentrations of the 14 furanocoumarins are determined in the three apiaceous vegetable types by analyzing the LC × LC chromatograms with MCR-ALS and manual integration for peak area determination. The concentrations of the analytes are found to vary greatly between samples, even within a single vegetable type.

Structures of Two Yellow Dyes Found in Cucumbers Pickled in Soy Sauce Colored with Tartrazine (Y4).

Two yellow dyes, together with tartrazine (Y4), were found in cucumbers pickled in soy sauce, for which the use of tartrazine is permitted, by TLC, LC-DAD, and LC-MS. The retention times on LC chromatograms and the maximum absorbance wavelengths measured by LC-DAD of the two dyes were different from those of tartrazine. Mass spectra of the dyes indicated that these dyes lacked one sulfonyl group of tartrazine. The presence of two less sulfonated dyes in tartrazine has been reported. Hence, the two less sulfonated dyes were synthesized. The two dyes found in cucumbers were compared with the synthesized dyes by LC-DAD and LC-MS. Since the retention times of the dyes in cucumbers on the LC chromatograms, as well as their LC-DAD spectra and mass spectra, were found to be identical with those of the synthesized dyes, we concluded these dyes are the less sulfonated subsidiary dyes of tartrazine.

Program for the interpretive optimization of two-dimensional resolution

The challenge of fully optimizing LC×LC separations is horrendous. Yet, it is essential to address this challenge if sophisticated LC×LC instruments are to be utilized to their full potential in an efficient manner. Currently, lengthy method development is a major obstacle to the proliferation of the technique, especially in industry. A program was developed for the rigorous optimization of LC×LC separations, using gradient-elution in both dimensions. The program establishes two linear retention models (one for each dimension) based on just two LC×LC experiments. It predicts LC×LC chromatograms using a simple van-Deemter model to generalize band-broadening. Various objectives (analysis time, resolution, orthogonality) can be implemented in a Pareto-optimization framework to establish the optimal conditions. The program was successfully applied to a separation of a complex mixture of 54 aged, authentic synthetic dyestuffs, separated by ion-exchange chromatography and ion pair chromatography. The main limitation experienced was the retention-time stability in the first (ion-exchange) dimension. Using the PIOTR program LC×LC method development can be greatly accelerated, typically from a few months to a few days.

Rapid Stability-indicating RRLC Method for Simultaneous Estimation of Irbesartan and its Related Impurities

A green, novel gradient stability-indicating reverse phase rapid resolution liquid chromatographic method was developed and validated for simultaneous estimation of irbesartan and along with six related impurities in active pharmaceutical ingredient samples. The chromatographic separation was achieved on Kromasil C8 (3.5 µm, 150×4.6 mm) short column with 0.1% v/v ortho-phosphoric acid and acetonitrile as mobile phase using gradient elution. The developed method showed good resolution between irbesartan and its six related impurities and were eluted within 15 min. run time of LC chromatogram. Regression analyses indicate correlation coefficient value greater than 0.999 for irbesartan and its six related impurities. The limit of detection for irbesartan and the known related impurities were observed at a level below 0.004% (0.019 µg/ml and the method is showing better recoveries for irbesartan (99.6–100.7%) and also for its six known impurities (88.5–98.9%). The test solution and related substances were found to be stable in the diluents for 24 h. The developed stability-indicating method is found to be rapid, accurate, precise, linear, specific, sensitive, rugged, robust, and stability-indicating. The application of developed method was also verified by an assay of irbesartan and related substances in commercial API bulk drug samples and more essentially, the method is economic and environment friendly than the other published methods.

Using the liquid-chromatographic-fingerprint of sterols fraction to discriminate virgin olive from other edible oils

A method to discriminate virgin olive oil from other edible vegetable oils such as, sunflower, pomace olive, rapeseed, canola, corn and soybean, applying chemometric techniques to the liquid chromatographic representative fingerprint of sterols fraction, is proposed. After a pre-treatment of the LC chromatogram data – including baseline correction, smoothing signal and mean centering – different unsupervised and supervised pattern recognition procedures, such as principal component analysis (PCA), hierarchical cluster analysis (HCA), and partial least squares-discriminant analysis (PLSDA), have been applied. From the information obtained from PCA and HCA, two groups can be clearly distinguished (virgin olive and the rest of vegetable oils tested) which have been used to discriminate between two defined classes by means of a PLSDA model. Five latent variables (LVs) explained 76.88% of X-block variance and 95.47% of the defined classes block (γ-block) variance. A root mean square error for calibration and cross validation of 0.10 and 0.22 respectively, confirmed these results and a root mean square error for prediction of 0.15 evidences that the classification model proposed presents an adequate prediction capability. The contingency table also shows the good performance of the model, proving the capability of the LC-R-FpM, to discriminate virgin olive from other vegetable edible oils.

Impact of reversed phase column pairs in comprehensive two-dimensional liquid chromatography

A major issue in optimizing the resolving power of two-dimensional chromatographic separations is the choice of the two phases so as to maximize the distribution of the analytes over the separation space. In this work, we studied the choice of appropriate reversed phases to use in on-line comprehensive two-dimensional liquid chromatography (LC×LC). A set of four chemically different conventional bonded reversed phases was used in the first dimension. The second dimension column was either a conventional bonded C18 phase or a carbon-clad phase (CCP). The LC×LC chromatograms and contour plots were all rather similar indicating that the selectivities of the two phases were also similar regardless of the reverse phase column used in the first dimension. Further, the spatial coverage seen with all four first dimension stationary phases when paired with a second dimension C18 phase were low and the retention times were strongly correlated. However, when the C18 column was replaced with the CCP column much improved separations were observed with higher spatial coverages, greater orthogonalities and significant increases in the number of observed peaks.

Peptides Quantification by Liquid Chromatography with Matrix-Assisted Laser Desorption/Ionization and Selected Reaction Monitoring Detection

We present a novel analytical platform for peptides quantitative assays in biological matrices based on microscale liquid chromatography fractionation and matrix-assisted laser desorption/ionization mass spectrometric detection using the selected reaction monitoring (SRM) mode. The MALDI source was equipped with a high frequency Nd:YAG laser (1000 Hz) and mounted on a triple quadrupole/linear ion trap mass spectrometer (MALDI-QqQ(LIT)). Compared to conventional LC-ESI-SRM/MS, the separated analytes are "time-frozen" onto the MALDI plate in fractions, and navigation through the LC chromatogram makes it possible to perform SRM experiments as well as enhanced product ion spectra acquisition for confirmatory analyses without time constraints. The LC spots were analyzed using different rastering speeds ranging from 0.25 to 4 mm/sec with the shortest analysis time of 425 ms/spot. Since the LC runs can be multiplexed and do not need a comprehensive investigation, the present platform offers a valuable alternative to LC-ESI-SRM/MS for high throughput proteomic analyses. In addition, the derivatization of the N-terminal α-amino group by sulfonation was found to be key for the fragmentation of singly charged peptides under low collision energy regime. Under such conditions, y-ion series were observed in the MS/MS spectra, and thus the design of SRM experiments was greatly simplified. The quantitative performance of the platform was compared to that of LC-ESI-SRM/MS by spiking yeast tryptic peptides in human plasma digests. Both platforms exhibited similar sensitivities, accuracy (within ±20%) and precision (under 20%) in the relative quantification mode. As a proof of principle, the relative and absolute quantification of proteins associated with glycolysis, glyoxylate and tricarboxylic acid (TCA) cycles over a growth time course of Saccharomyces cerevisiae on glucose media was successfully performed using isotopic dilution.