Reversed-phase High-performance Liquid Chromatographic Separation Research Articles

Novel Green Chromatographic Approaches for Estimation of a Triple Common Cold Pharmaceutical Combination.

Nowadays, there is a strong interest in the scientific community in developing innovative methodologies within a green analytical chemistry framework. Herein, we introduce the first chromatographic approaches for the concurrent estimation of paracetamol (PAR), carbinoxamine (CRX), and pseudoephedrine (PSE) intended to relieve COVID-19 and common cold symptoms. The first method was thin layer chromatography (TLC) densitometry, which depends on the separation of the studied medications on TLC silica gel plates using ethyl acetate: methanol: ammonia (7.0: 3.0: 0.2, by volume) as the developing system, and were scanned at 208.0nm. The data were linear in the ranges of 1-25μg/band for PAR, 1-25μg/band for PSE and 0.1-5μg/band for CRX. The second method was reversed-phase high-performance liquid chromatography separation on a Kromasil C18 column using a mixture of 0.01M phosphate buffer containing 0.1% triethylamine (pH3.5) adjusted with orthophosphoric acid and ethanol at a flow rate of 1.0 mL/min in a gradient program. The separated peaks were detected at 215.0 nm over a concentration range of 10-250μg/mL for PAR, 5-35μg/mL for PSE, and 0.5-25μg/mL for CRX. Both approaches were validated according to International Conference on Harmonization (ICH) guidelines. Finally, the impact of these methods on the environment was evaluated by many tools.

Antioxidant peptides generated from chicken feet protein hydrolysates.

As major industrial poultry by-products, chicken feet are considered as notable sources of several bioactive molecules. The current work covers the processing of chicken feet proteins as substrates to be hydrolysed by combinations of three commercial enzymes (Alcalase®, Flavourzyme® and Protana® Prime) during different hydrolysis periods and the evaluation of the identified peptides having antioxidant activity after simulated gastrointestinal digestion. Enzymatic hydrolysis with Alcalase® and Protana® Prime combination for 4 h resulted in the highest activities. Reversed-phase high-performance liquid chromatographic separation of the purified hydrolysate yielded three active fractions that were further identified by nano-liquid chromatography-tandem mass spectrometry. The bioactivities of over 230 identified peptide sequences were estimated after simulated gastrointestinal digestion, and those peptides with the highest chance of exerting antioxidant activity were selected to be further synthesised and tested. In this sense, the synthesised dipeptides CF and GY showed the highest antioxidant capacity. CF presented IC50 values of 69.63 and 145.41 μmol L-1 in 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and oxygen radical absorbance capacity (ORAC) assays, respectively. In contrast, GY IC50 values were 15.27 and 10.06 μmol L-1 in ABTS and ORAC assays, respectively. Significant differences (P < 0.05) were registered between peptides in the same antioxidant assays. Overall, the findings emphasised the favourable impact of enzymatic hydrolysis with the obtaining of antioxidant peptides from poultry by-products that could be evaluated as a safe and economical source to retard oxidation in food systems. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Determination of rFVII Concentration in Cell Culture Supernatant Using VIISelect Resin and RP-HPLC-UV

Background: Recombinant activated human coagulation factor VIIa (rFVIIa), a vitamin K-dependent serine protease, was originally developed by Novo Nordisk® for the treatment of patients with FVII deficiency as well as patients with hemophilia A and B with inhibitors against FVIII or FIX. The gene for human FVII is cloned and expressed in baby hamster kidney (BHK) cells, and the protein is secreted into the culture media, which is then converted to the active form during chromatographic purification steps. When secreted into the culture media, measuring the concentration levels of FVII is needed in order to monitor, control and optimize the production processes. However, because of the high complexity of such media, reliable analytical techniques are required for this purpose. Objective: This work focuses on the analytical application of VIISelect resin (GE Healthcare) as a highly selective adsorbent for cleanup and preconcentration of rFVII in culture supernatant before analysis by reversed-phase high-performance liquid chromatography (RP-HPLC). Method: Empty 1 ml SPE cartridges were packed with VIISelect resin. Four types of solutions were used for the sample preparation. The RP-HPLC separation was conducted on a C4 column with UV detection. Results: The method shows recoveries greater than 97% in culture medium with relative standard deviations (RSDs) of less than 2%. The limits of detection and quantification were 0.039 mg/L and 0.12 mg/L, respectively. Conclusion: The method showed better performance in terms of precision and accuracy for rFVIIa determination in cell culture supernatant compared to other techniques, like ELISA and SDSPAGE.

Efficient separation and quantification of methyl palmitate and methyl oleate in biodiesel mixture using reverse-phase high performance liquid chromatography

ABSTRACT The excessive utilization of petroleum resources leads to global warming, crude oil price fluctuations, and fast depletion of petroleum reserves. Biodiesel (Fatty Acid Methyl Ester, FAME) has gained importance as a clean and renewable energy source over the last few years. It is produced by reacting vegetable oils and short-chain alcohols via transesterification/esterification reaction. The properties of biodiesel depend on the vegetable oils’ fatty acid composition due to the saturated and unsaturated fatty acid content variation. During the FAME quantification analysis, separation of methyl palmitate (MeP) and methyl oleate (MeO) are major challenges in the C18 column in High Performance Liquid Chromatography (HPLC). These components are difficult to quantify as they elute simultaneously by forming an overlapping peak. A new reverse-phase HPLC separation method with C18 column has been developed to efficiently separate and quantify FAMEs derived from palm oil. UV-VIS detector was used at 205 nm. The FAMEs separation was achieved using ternary mobile phase solvents acetonitrile, water, and acetone with the composition of 62%, 33%, and 5% in an isocratic flow with 2.20 mL/min flowrate and 45 °C column temperature. Methyl esters were analyzed with satisfactory linearity (r 2 >0.99), repeatability (relative standard deviation <2%), and sensitivity (limit of quantification).

Molecular diversification of antimicrobial peptides from the wolf spider Lycosa sinensis venom based on peptidomic, transcriptomic, and bioinformatic analyses.

The venom of Lycosoidea spiders is a complex multicomponent mixture of neurotoxic peptides (main components) and antimicrobial peptides (AMPs) as minor components. In this study, we described the high-throughput identification and analysis of AMPs from Lycosa sinensis venom (named LS-AMPs) using a combination strategy that includes the following three different analysis approaches: (i) peptidomic analysis, namely reversed-phase high-performance liquid chromatography (RP-HPLC) separation plus top-down sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS); (ii) transcriptomic analysis, namely cDNA library construction plus DNA sequencing; (iii) bioinformatic analysis, namely analysis and prediction for molecular characters of LS-AMPs by the online biology databases. In total, 52 sequences of AMPs were identified from L. sinensis venom, and all AMPs can be categorized into eight different families according to phylogenetic analysis and sequence identity. This is the largest number of AMPs identified from a spider species so far. In the present study, we demonstrated molecular characteristics, such as complex precursor, N- and/or C-terminally truncated analogs, and C-terminal amidation of LS-AMPs from L. sinensis venom. This is a preliminary investigation on the molecular diversification of venom-derived AMPs from the wolf spider species (family Lycosidae), and a detailed investigation on the functional diversity of LS-AMPs will be preformed in the future.

RP-HPLC AND CHEMOMETRIC METHODS FOR THE DETERMINATION OF TWO ANTI-DIABETIC MIXTURES; METFORMIN HYDROCHLORIDE-CANAGLIFLOZIN AND METFORMIN HYDROCHLORIDE-GLICLAZIDE IN THEIR PHARMACEUTICAL FORMULATION

Objective: To develop and validate novel more sensitive analytical methods for the concurrent quantification of metformin-canagliflozin and metformin-gliclazide in their bulk forms and in their pharmaceutical preparations.&#x0D; Methods: Two methods were developed based on several chemometric assisted spectrophotometric methods and a Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC). The first method applies different spectrophotometric chemometric assisted methods, including ratio difference, derivative ratio and extended ratio subtraction method, while the second method describes a RP-HPLC separation of metformin hydrochloride-canagliflozin and metformin hydrochloride-gliclazide binary mixtures using a C18 column with a mobile phase consisting of acetonitrile: potassium dihydrogen phosphate (adjusted to pH 3) with sodium lauryl sulphate as additive in the ratio of 30:70 (%v/v) in isocratic elution mode at 1 ml/min.&#x0D; Results: The proposed methods were able to quantify each of the studied drugs in their binary mixtures with high percentage recoveries in both methods. The spectrophotometric methods were able to quantify each of metformin, canagliflozin and gliclazide in the ranges of 2.0-20.0 μg/ml, 1.5-40.0 μg/ml and 2.0-30.0 μg/ml, respectively. The RP-HPLC method produced well-resolved peaks at a retention time of 3.92, 6.92 and 9.10 min in the concentration ranges of 50.0-300.0 μg/ml, 5.0-50.0 μg/ml and 10.0-100.0 μg/ml for metformin, canagliflozin and gliclazide, respectively. The proposed methods were optimized and validated in accordance to the International Conference of Harmonisation (ICH) guidelines in terms of linearity, LOD, LOQ, precision and accuracy.&#x0D; Conclusion: The developed methods were found to be sensitive and reproducible methods for the simultaneous determination of anti-diabetic binary mixtures; metformin hydrochloride-canagliflozin and metformin hydrochloride-gliclazide. And thus were successfully employed for the quality control analysis of the pharmaceutical formulations of the studied binary mixtures.

Response Surface Methodology used in the Optimization of RP-HPLC Condition for Quantitative Analysis of Carmine and Rhodamine B

The objective of this study was to optimize reversed-phase high-performance liquid chromatography (RP-HPLC) using an experimental design approach based on the response surface methodology of Central Composite Design (CCD) for separation and analysis of carmine (CAR) and Rhodamine B (RHO) in lipstick products. Some factors (independent variables) responsible for RP-HPLC separation including pH of buffer phosphate (X1), the acetonitrile ratio (X2), flow rate of mobile phase (X3), and column temperature (X4) were investigated. While, the responses (dependent variables) evaluated were resolution between CAR and RHO (Y1), tailing factor of CAR (Y2), tailing factor of RHO (Y3), retention time of CAR (Y4), retention time of RHO (Y5), peak area of CAR (Y6) and peak area of RHO (Y7). CCD showed that separation of CAR and RHO was influenced by these independent variables (factors). The optimum predicted conditions for the separation of CAR and RHO based on statistical results was pH buffer of 3.4, ACN 55%, the flow rate of 1.1 mL/min and column temperature of 35 o C with the desirability of 1. Both CAR and RHO were clearly separated using optimum conditions, as suggested by CCD. The developed techniques were effective for optimizing chromatographic separation, therefore, the time consumption and a large number of running could be hindered.

CENTRAL COMPOSITE DESIGN APPLIED IN HPLC OPTIMIZATION FOR ANALYSIS OF TARTRAZINE AND AURAMINE O IN POWDER DRINKS

Objective: This study was intended to optimize reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determination of Tartrazine (TAR) and Auramin O (AUO) in powder drinks using experimental design of central composite design (CCD) approach.&#x0D; Methods: TAR and AUO in powder drink product has same properties, therefore both analytes were analysed using C18 column (XBridge Shield RP 18 250 mm x 4.6 mm i.d., 5 µm) using Shimadzu LC 20AD chromatograph equipped with photo-diode array (PDA) detector at 300-650 nm. Some factors responsible for RP-HPLC separation of TAR and AUO including the concentration of buffer, the ratio of mobile phase and flow rate were optimized using CCD. The responses evaluated were peak area, retention time, and tailing factor. The mobile phase used was acetonitrile and ammonium acetate buffer, and acetonitrile composition was optimized at 84-86% for separation of TAR and AUO, delivered at a flow rate of 0.8–1.2 ml/min, using ammonium acetate buffer at 19-21 mmol.&#x0D; Results: CCD showed that separation of TAR and AUO was influenced by flow rate, the ratio of acetonitrile and ammonium acetate concentration. These factors affected significantly to retention time, peak area, and tailing factor. The optimal condition obtained based on CCD was flow rate of 1.2 ml/min, the ratio of acetonitrile 86%, and ammonium acetate concentration of 19 mmol.&#x0D; Conclusion: CCD can be used to get optimum condition for analysis of TAR and AUO in powder drink product.

Detection of multiclass explosives and related compounds in soil and water by liquid chromatography-dielectric barrier discharge ionization-mass spectrometry.

In this work, the multiclass detection of explosives and related compounds has been studied by liquid chromatography-high-resolution mass spectrometry by means of a time-of-flight mass spectrometer (TOFMS) operated in the negative ion mode, using dielectric barrier discharge ionization (DBDI). Reversed-phase high-performance liquid chromatography (HPLC) separation was performed using water-methanol mobile phase without any modifier, although the effect of ammonium acetate was studied. DBD plasma was generated by applying a square wave voltage of 2.5kV to a 100-mLmin-1 He flow. The DBDI probe was adjusted to fit the commercial API source housing so that the HPLC eluent was nebulized and vaporized in the same manner as for atmospheric-pressure chemical ionization (APCI). The ionization process was affected by the temperature of the two nitrogen streams used to vaporize the solvent and the analytes, particularly for RDX and HMX, which are thermolabile compounds. The best approach in terms of sensitivity for all the studied compounds was the use of a gradient of temperatures in the ionization source, starting at 225/200°C (vaporizer/drying gas temp) for nitramines and ending at 350/325°C for nitroaromatic compounds. High-resolution full-scan spectra of individual selected compounds were recorded by LC-DBDI-TOFMS, and the results were compared to LC-APCI-TOFMS. A better sensitivity (slope of calibration curve) was obtained by DBDI for more than 70% of the studied compounds in both wastewater and soil extracts. Graphical abstract ᅟ.

Simultaneous determination of total homocysteine, cysteine, glutathione, and N-acetylcysteine in brain homogenates by HPLC.

We have developed a simple, fast, accurate, and cheap method for the simultaneous determination of total cysteine, homocysteine, glutathione, and N-acetylcysteine in brain homogenates based on the reduction of disulfide bonds by tris(2-carboxyethyl) phosphine, pre-column derivatization of free thiol groups with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pair reversed-phase high-performance liquid chromatography separation with ultraviolet detection. The separation of thiol derivatives was achieved in 10min. Linearity was observed in the range of 10-300, 0.7-10, 2-30, and 3-20μmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2μmol/L homogenate for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The precision, calculated as relative standard deviation, was in the range of 1.21-4.77, 1.53-14.35, 0.47-1.92, and 1.61-8.95% for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The presented method was successfully applied to the selective determination of total amino thiols in pig brain tissue samples.

Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization

Free thiol content, and its consistency, is one of the product quality attributes of interest during technical development of manufactured recombinant monoclonal antibodies (mAbs). We describe a new, mid/high-throughput reversed-phase-high performance liquid chromatography (RP-HPLC) method coupled with derivatization of free thiols, for the determination of total free thiol content in an E. coli-expressed therapeutic monovalent monoclonal antibody mAb1. Initial selection of the derivatization reagent used an hydrophobicity-tailored approach. Maleimide-based thiol-reactive reagents with varying degrees of hydrophobicity were assessed to identify and select one that provided adequate chromatographic resolution and robust quantitation of free thiol-containing mAb1 forms. The method relies on covalent derivatization of free thiols in denatured mAb1 with N-tert-butylmaleimide (NtBM) label, followed by RP-HPLC separation with UV-based quantitation of native (disulfide containing) and labeled (free thiol containing) forms. The method demonstrated good specificity, precision, linearity, accuracy and robustness. Accuracy of the method, for samples with a wide range of free thiol content, was demonstrated using admixtures as well as by comparison to an orthogonal LC-MS peptide mapping method with isotope tagging of free thiols. The developed method has a facile workflow which fits well into both R&D characterization and quality control (QC) testing environments. The hydrophobicity-tailored approach to the selection of free thiol derivatization reagent is easily applied to the rapid development of free thiol quantitation methods for full-length recombinant antibodies.

Development and validation of stability indicating liquid chromatographic (RP-HPLC) method for estimation of ubidecarenone in bulk drug and formulations using quality by design (QBD) approach

A novel, accurate, precise and economical stability indicating Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method, was developed and validated for the quantitative determination of ubidecarenone (UDC) in bulk drug, UDC marketed formulation and UDC loaded cubosomes (CBMs) nanocarriers through Response surface methodology (RSM) design with three factors and three levels was performed to optimize the chromatographic variables followed by forced degradation studies of UDC were performed to detect degradation peak. RP-HPLC separation was achieved using mobile phase consisting of Acetonitrile:Tetrahydrofuran:Deionised water in the ratio 55:42:3 and a flow rate of 1.0 mL/min was optimized with a standard retention time (Rt) of 2.15 min, through experiment. The method was found linear in the concentration range of 5-100 µg/mL with a regression coefficient of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 3.04 µg/mL and 9.11 µg/mL, respectively.

A METHOD FOR DETERMINING 1,4-BENZOTHIAZINE DERIVATIVES IN RAT PLASMA BY HPLC AND ITS APPLICATION TO A PHARMACOKINETIC STUDY

Objective: The objective of the study was to develop, optimize and validate of a new reverse-phase high-performance liquid chromatography (RP-HPLC) method for the determining 1,4-benzothiazine derivatives (AR13 and AR15) in a biological sample of rat plasma. The 1,4-benzothiazine derivatives are produced by the synthetic reactions.Methods: RP-HPLC separation was performed using an ODS-2 Hypersil column with gradient elution mobile phase consisting of water-acetonitrile for AR13 and AR15 (1:9 v/v, 3:7 v/v) at room temperature 1 ml/min flow rate, and interfaced with photodiode array detector (PDA) detector, 233 nm, 235 nm respectively.Results: A linear response was obtained between (range from 0.100-10.00 mg/ml) AR13 and (range from 0.096–9.88 mg/ml) AR15 with correlation coefficient 0.999 and 0.998. The linearity range of both AR13 and AR15 was 101.65±1.5 and 98.78±1.7.Conclusion: It was concluded that the method was simple, accurate, sensitive, accurate and reproducible and has been successfully applied to the pharmacokinetic study of AR13 and AR15 in rat plasma.

Determination and Degradation Study of Enalapril Maleate by High Performance Liquid Chromatography

Enalapril maleate belongs to the group angiotensin-converting enzyme inhibitors used in the treatment of hypertension and heart failure. In this work, reversed-phase high-performance liquid chromatography (RP-HPLC) method for the determining the level of impurities in enalapril maleate tablets was developed and validated. A Snyder triangle was approved for the optimization of RP-HPLC separation of enalapril in the presence of five impurities and two degradation products. Separation was achieved on an octadecyl silica column using a mobile phase of 20 mM phosphate buffer (pH 2) – methanol – THF – TEA (66 : 25 : 9 : 0.1, v/v) with UV detection at 215 nm. The degree of linearity and the characteristic statistical parameters of calibration curves, including the limit of detection (LOD) and limit of quantitation (LOQ), for Enalapril maleate and its degradation products were estimated. The method was found to be specific, accurate, and precise. The stability indicating capability of the assay was proved by conducting a test under forced degradation conditions of temperature and humidity on a commercial drug product.

Liquid chromatographic enantioseparation of carbocyclic β-amino acids possessing limonene skeleton on macrocyclic glycopeptide-based chiral stationary phases

Polar-ionic and reversed-phase high-performance liquid chromatographic separations of limonene-based cyclic β-amino acid enantiomers were carried out by using macrocyclic glycopeptide-based chiral selectors applying Chirobiotic T, TAG and R columns. The effects of additives, concentration of the co- and counter-ions and the temperature in polar-ionic mobile phase systems were studied. The influence of pH, MeOH content and alcohol additives were investigated in the reversed-phase mode. The difference in the change in standard enthalpy Δ(ΔH°), entropy Δ(ΔS°), and free energy Δ(ΔG°) was calculated from the linear van't Hoff plots derived from the ln α vs 1/T curves in the temperature range 5–40°C. Unusual temperature behavior was observed on Chirobiotic TAG for most of the analytes: decreased retention times were accompanied with increased separation factors with increasing temperature, and separation was entropically-driven. For two of the studied analytes enthalpically-driven enantioseparations were observed. The elution sequence was determined in all cases, but no general rule could be established.

Fast Fatty Acid Analysis by Core-Shell Reversed-Phase Liquid Chromatography Coupled to Evaporative Light-Scattering Detector

A high-performance liquid chromatography analysis method with an evaporative light-scattering detector has been developed for the separation and quantitative analysis of free fatty acids in biological matrices. Core-shell reversed-phase high-performance liquid chromatography separation of 10 free fatty acids is achieved within 10.5 min using a methanol/water (0.05% trifluoroacetic acid) eluent gradient. After optimization, the drift tube and nebulization temperature of the evaporative light-scattering detector was set at 35°C, nitrogen flow-rate at 1.1 standard liter per minute and column temperature at 25°C. All calibration curves showed good regression (r2 > 0.9975). A validation procedure following the International Conference on Harmonisation guidelines was implemented to certify the method. Relative standard deviations did not exceed 1.5% and 4.25% for repeatability and reproducibility respectively.

STUDIES ON HPTLC AND RP-HPLC METHODS FOR DETERMINATION OF TADALAFIL IN PHARMACEUTICAL DOSAGE FORM

Tadalafil is a selective phosphodiesterase Type 5 enzyme inhibitor. Simple and sensitive High Performance Thin - Layer Chromatography (HPTLC) and Reversed - Phase High - Performance Liquid Chromatography (RP-HPLC) methods have been developed for estimation of tadalafil in pharmaceutical dosage form. Separation of tadalafil was achieved on HPTLC aluminum sheets of silica gel 60 F254 with mixture of toluene: methanol (7.2: 2.8 V/V) as the mobile phase. tadalafil obeyed linearity in the concentration range of 300 - 800 ng/band with correlation coefficient (r2) 0.9994. Detection was performed densitometricaly at 284 nm with Rf 0.50 ± 0.02. RP-HPLC separation was achieved using Qualisil C18 column on mixture of methanol: water (75: 25 % V/V) as the mobile phase. Linearity was plotted in range of 2 – 12 μg/mL and (r2) value as 0.9998. The retention time was found to be 4.82 ± 0.02 min. The developed HPTLC and RP-HPLC methods have been successfully applied for the determination of tadalafil in bulk and pharmaceutical formulation. Both these methods were statistically compared.

Reversed-phase high-performance liquid chromatographic separation of some 2-arylpropionic acids using vancomycin as chiral stationary phase

A rapid, sensitive and reproducible HPLC method has been developed for enantioseparation of six non-steroidal anti-inflammatory drugs, which are acidic compounds: carprofen, fenoprofen, flurbiprofen, ibuprofen, indoprofen and ketoprofen. The effects of the mobile phase composition on retention times and resolutions of the analytes were studied. A column based on vancomycin immobilized by reductive amination to aldehyde functionalised silica was prepared in house and used. The prepared sorbent shows a great stability and selectivity over a range of pH (4–6), and the separation was carried out using the mobile phase composed of a mixture of 40 % of methanol in ammonium nitrate buffer (50 mM) at pH 5.0. Another mobile phase consisted of 50 % of methanol in phosphate buffer (5 mM) at pH 5.0 was also prepared and tested. The two mobile phases are the optimum conditions obtained. All experiments were conducted at flow rate 0.6 ml/min, using a UV detector wavelength at λ = 254 nm.

The complexity and structural diversity of ant venom peptidomes is revealed by mass spectrometry profiling.

Compared with other animal venoms, ant venoms remain little explored. Ants have evolved complex venoms to rapidly immobilize arthropod prey and to protect their colonies from predators and pathogens. Many ants have retained peptide-rich venoms that are similar to those of other arthropod groups. With the goal of conducting a broad and comprehensive survey of ant venom peptide diversity, we investigated the peptide composition of venoms from 82 stinging ant species from nine subfamilies using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). We also conducted an in-depth investigation of eight venoms using reversed-phase high-performance liquid chromatography (RP-HPLC) separation coupled with offline MALDI-TOFMS. Our results reveal that the peptide compositions of ant venom peptidomes from both poneroid and formicoid ant clades comprise hundreds of small peptides (<4 kDa), while large peptides (>4 kDa) are also present in the venom of formicoids. Chemical reduction revealed the presence of disulfide-linked peptides in most ant subfamilies, including peptides structured by one, two or three disulfide bonds as well as dimeric peptides reticulated by three disulfide bonds. The biochemical complexity of ant venoms, associated with an enormous ecological and taxonomic diversity, suggests that stinging ant venoms constitute a promising source of bioactive molecules that could be exploited in the search for novel drug and biopesticide leads.

Precolumn o‐Phthalaldehyde‐N‐acetyl‐L‐cysteine Derivatization Followed by RP‐HPLC Separation and Fluorescence Detection of Sitagliptin Enantiomers in Rat Plasma

An indirect reversed-phase high-performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o-phthalaldehyde and N-acetyl-L-cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP-18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50-5000 ng/ mL for both enantiomers. The intra- and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments.