Simple Pre-column Derivatization Research Articles

In-silico Toxicity Assessment and Trace Level Quantification of Veratryl Chloride a Potential Genotoxic Impurity in Ivabradine Hydrochloride using LC-MS/MS

A sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) mode was developed and validated for trace analysis of 4-(chloromethyl)-1, 2-dimethoxybenzene (Veratryl chloride) a potential genotoxic impurity in Ivabradine hydrochloride (IVB). Chromatographic separation was performed on a Poroshell 120EC C18 (50 × 3.0 mm, 2.7 μm) column using a mixture of 10 mM ammonium formate and acetonitrile in isocratic elution mode at a 0.25 mL/min flow rate. A simple pre-column derivatization with di-ethylamine was employed for the derivatization of the veratryl chloride. The developed LC-MS/MS method was linear and accurate in the 1.5–10.0 ppm concentration range with r2 ˃ 0.999 and percent recoveries greater than 90%. The developed method was precise with RSD (%) of not more than 4.5%. In-silico genotoxicity and carcinogenicity potential of veratryl chloride was assessed using ICH M7 principles found to be positive. The developed method can identify and quantify veratryl chloride in IVB, hence can be applied by quality control labs of pharmaceutical industries for trace quantification.

Determination of Total Proteinogenic Amino Acids and Taurine by Pre-column Derivatization and UHPLC: Single Laboratory Validation, First Action Official MethodSM 2019.09.

A method has been developed and validated for selective, accurate, and precise determination of total proteinogenic amino acids and taurine from infant formula and adult/pediatric nutritional formulas (powders, ready-to-feed liquids, and liquid concentrates). The method was reviewed by the AOAC INTERNATIONAL SPIFAN Expert Review Panel (ERP) during the 133rd AOAC Annual Meeting and Expo on September 7, 2019 in Denver, CO, USA, and was recommended to First Action Official MethodsSM status. The method involves protein hydrolysis to amino acids, a simple pre-column derivatization of amino acids and separation of derivatized amino acids by UHPLC. The quantification of amino acids is performed by multi-point calibration using norvaline as the internal standard. The analytical method is capable of quantitative determination for 22 proteinogenic amino acids, but cannot be used to quantitate tryptophan, which is destroyed during the acid hydrolysis step. Asparagine is determined as aspartic acid and glutamine as glutamic acid. Cystine and cysteine are converted to S-2-carboxyethylthiocysteine (CYSx) and the derivative is separated from the other amino acids. Citrulline, which is present in some matrixes, is separated from other amino acids and is not included in the method performance evaluation in the single-laboratory validation (SLV). The proposed method met all the performance requirement limits set in Standard Method Performance Requirement (SMPR®) 2014.013 for total proteinogenic amino acids and taurine. The correlation coefficient of multi-point calibration was not less than 0.999 for any amino acids at any point in the SLV study, confirming the validity of linear dynamic range (LDR) and linearity of the method. The individual amino acids in the chromatogram were identified by absolute retention time and relative retention time (RRT) with respect to the internal standard norvaline. There were no significant (S/N <10) interferences from the reagents or byproducts of derivatization and targeted matrixes. The method demonstrated high selectivity. Accuracy of the method was validated using standard reference materials (SRMs; NIST SRM 1869 and 1849a) and spike recovery studies. The amino acid results in the SRMs were within the ranges of Reference Mass Fraction Values. The accuracy of the method was corroboratively validated by spike recovery studies. The average spike recovery ranged between 93 and 107% ensuring the accuracy of the method for amino acids and compliance with AOAC SMPR 2014.013. Precision data of the method demonstrate that it meets the stakeholder requirements as per the SMPR. The mean RSDr for all the amino acids for 17 matrixes selected for the SLV were not more than 4%. The method is very sensitive and the LOQ can go down to approximately ten times lower than the SMPR requirements. The sensitivity of method is a direct reflection of its signal-to-noise ratio which ensures guaranteed method performance at the lower levels of amino acids in these matrixes. Taurine (aminoethane sulfonic acid), unlike the other amino acids, is a beta-sulfonic amino acid that is not used in protein synthesis but is found as a free amino acid in tissues. The acidic functional group (-COOH) in common amino acids is replaced with a sulfonic acid (-SO3H) group in taurine. The method offers baseline separation of citrulline which is an alpha amino acid generally present in infant formula and adult/pediatric nutritional products. The separation of citrulline eliminates the risk of interference of this compound with other amino acids. The method can also separate and quantitate hydroxyproline, an important component of collagen that is often used to quantitate collagen. The method is simple and does not include any proprietary chemicals or instruments, and it can be performed on any basic reverse phase UHPLC system with UV detection (1-6).

Simultaneous screening of homotaurine and taurine in marine macro-algae using liquid chromatography–fluorescence detection

Simultaneous analysis of homotaurine and its homologous, taurine, is a highly challenging issue, especially in matrices they exist simultaneously. A simple precolumn derivatization procedure combined with high-performance liquid chromatography–fluorescence detection was developed for simultaneous determination of homotaurine and taurine in marine macro-algae. The analytes were derivated with o-phthalaldehyde at an ambient temperature and alkaline medium. Calibration curves were linear in the ranges of 50–2500 µg L−1 for homotaurine and 100–2500 µg L−1 for taurine with the coefficients of determination higher than 0.998. Limits of detection of homotaurine and taurine were 15 and 30 µg L−1, respectively. Intraday (n = 6) and inter-day (n = 4) precisions of the method were satisfactory with relative standard deviations less than 6.0%. Good recoveries (>94%) were acquired by the method for extraction of homotaurine and taurine from algae matrices. Liquid chromatography–mass spectrometry was also used to confirm detection of the analytes in algae samples. The data suggest that the method was successfully applied to simultaneous determination of homotaurine and taurine in algae samples.

Determination of residual dimethyl sulfate in hexaconazole technical (fungicide) by head space gas chromatography mass spectrometry

Dimethyl sulfate is used as alkylating agent in manufacturing process but because of its genotoxicity nature it should be monitored at trace levels (1mg/kg). A simple pre-column derivatization i.e. alkylation of butyl alcohol gives corresponding ether (methoxybutane) which is analyzed by head space gas chromatography mass spectrometry (HS-GC–MS). The retention time of derivative is 5.32min. Analysis was carried out in Selected Ion Monitoring (SIM) mode. The linear regression analysis data for the calibration curve showed a good linear relationship with a regression coefficient of 0.999 in the concentration range of 0.035mg/kg to 9.234mg/kg. The method was validated for specificity, linearity, LOD, LOQ, precision and accuracy.

A simple pre-column derivatization method for the determination of mancozeb technical (fungicide) by reverse phase HPLC-UV

A simple and fast pre-column derivatization high performance liquid chromatographic method for the technical analysis of mancozeb was developed and validated in the present study.

Trace determination of lenalidomide in plasma by non-extractive HPLC procedures with fluorescence detection after pre-column derivatization with fluorescamine

BackgroundLenalidomide (LND) is a new potent drug used for treatment of multiple myeloma. For its pharmacokinetic studies and therapeutic monitoring, a proper analytical method was required.ResultsIn this study, a non extractive and simple pre-column derivatization procedures have been proposed, for the for trace determination of lenalidomide (LND) in human plasma by HPLC with fluorescence detection. Plasma samples were treated with acetonitrile for protein precipitation then treated with copper acetate to form stable complexes with the biogenic amines and mask their interference with the derivatization reaction of LND. Treated plasma samples containing LND was derivatized with fluorescamine (FLC) in aqueous media at ambient temperature. Separation of the derivatized LND was performed on Hypersil BDS C18 column (250 × 4.6 mm, 5 μm particle size) using a mobile phase consisting of phosphate buffer (pH 4):methanol: tetrahydrofuran (70:10:20, v/v) at a flow rate of 1.0 mL/min. The derivatized samples were monitored at an emission wavelength of 495 nm after excitation at a wavelength of 382 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9997, n = 9) was found between the peak area and LND concentrations in the range of 2–100 ng/mL. The limits of detection and quantitation were 0.8 and 2.30 ng/mL, respectively. The intra- and inter-assay precisions were satisfactory and the accuracy of the method was proved. The recovery of LND from the spiked human plasma was 99.30 ± 2.88.ConclusionsThe proposed method had high throughput as the analysis involved simple sample pre-treatment procedure and a relatively short run-time (< 15 min). The results demonstrated that the method would have a great value when it is applied in the therapeutic monitoring and pharmacokinetic studies for LND.

Quantification of Glutathione in Plasma Samples by HPLC Using 4-Fluoro-7-nitrobenzofurazan as a Fluorescent Labeling Reagent

A rapid and highly sensitive high-performance liquid chromatograpy method with fluorescence detection has been developed for determination of glutathione (GSH) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized glutathione was performed using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)-acetonitrile (77:23, v/v) at a flow rate of 1.0 mL/min with the column temperature 2°C. The eluted derivatives were fluorometrically detected at an excitation wavelength 470 nm and an emission wavelength 530 nm. Under the optimum chromatographic conditions, the calibration curve was linear over the range of 0.1 µmol/L to 10.0 µmol/L with the correlation coefficient of 0.9988. The precision of the method was satisfactory with the intra- and inter-day coefficient of variation being 6.3%, 6.9%, respectively. This method has been used to determine glutathione concentrations in plasma samples from healthy individuals.

Improved Determination of d‐Glucosamine Hydrochloride in Health Foods by HPLC Using 7‐Fluoro‐4‐Nitrobenzo‐2‐Oxa‐l,3‐Diazole as a Derivative

This article presents an improved method to detect D-glucosamine hydrochloride in health foods. A simple precolumn derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. The separation of the derivatized D-glucosamine hydrochloride (NBD-D-glucosamine hydrochloride) was performed using a mobile phase consisting of acetonitrile, potassium dihydrogen phosphate (0.01 mol/L), and trifluoroacetic acid (350:649.74:0.26, volume ratio) at a flow rate of 1.0 mL/min with the column temperature 35 °C. Under the optimum chromatographic conditions, the peak area of NBD-D-glucosamine hydrochloride compared with its absolute value of the peak area of NBD-D-glucosamine hydrochloride in a standard solution concentration range from 1.0 to 500.0 mg/L showed a good linear calibration (R = 0.9999). Recoveries, at spiked concentrations of 10.0, 40.0, and 500.0 mg/L, varied between 97.2% and 102.6% with relative standard deviations ranging from 0.4% to 1.5%. The present method provides sufficient sensitivity as reflected by the values of limit of detection (LOD) and limit of quantification (LOQ). LOD was determined from the signal-to-noise ratios (S/N) of NBD-D-glucosamine hydrochloride peak of at least 3 in the recovery test at 0.02 mg/L, and the estimated LOQ was 0.06 mg/L (S/N = 10). The proposed method was successfully applicable to detect D-glucosamine hydrochloride in health foods and drugs containing a variety of complex materials.

Highly Sensitive LC Method with Automated Co-Sense System and Fluorescence Detection for Determination of Sertraline in Human Plasma

A highly sensitive LC method with column-switching “Co-sense” system and fluorescence detection has been proposed for trace determination of sertraline in human plasma. A simple pre-column derivatization procedure with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reagent was employed. Fluxetine was used as an internal standard. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient (r = 0.9997) was found between the peak area ratio and sertraline concentration in the range of 5–5,000 ng mL−1. The limit of detection and limit of quantitation were 1.41 and 4.28 ng mL−1, respectively. The intra- and inter-assay precisions were satisfactory; the relative standard deviations did not exceed 5.63%. The accuracy of the method was proved; the recovery of sertraline from the spiked human plasma was 99.76–102.62 ± 2.19–5.63%. The proposed method had high throughput as the analysis involved simple sample pre-treatment procedure and short run-time (~12 min). The results demonstrated that the method would have a great value if applied in bioavailability and pharmacokinetic studies for sertraline.

A highly sensitive HPLC method with automated on-line sample pre-treatment and fluorescence detection for determination of reboxetine in human plasma

A fully automated, rapid and highly sensitive HPLC method with automated sample pre-treatment by column-switching system and fluorescence detection has been developed for the trace quantitative determination of the new antidepressant reboxetine (RBX) in human plasma. A simple pre-column derivatization procedure with 7-flouro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) reagent was employed. Paroxetine (PXT) was used as an internal standard. Plasma samples containing both RBX and PXT, after filtration, were derivatized by heating with NBD-F in borate buffer of pH 8 at 70 °C for 30 min. The derivatized plasma samples were injected into the HPLC system where an on-line sample clean up was achieved on the pre-treatment column (Co-sense Shim-pack MAYI-ODS) with a washing mobile phase (acetonitrile:2% acetic acid; 40:60, v/v) at a flow rate of 5 mL min −1 for 1 min. After an automated on-line column switching to the analytical Hypersil phenyl 120A column (250 mm × 4.6 mm, 5 μm), the separation of the derivatized RBX and PXT was performed using a mobile phase consisting of sodium acetate buffer (pH 3.5):tetrahydrofuran:acetonitrile (55:35:10, v/v/v) at a flow rate of 2.0 mL min −1. The eluted derivatives were monitored by a fluorescence detector set at an excitation wavelength of 470 nm and an emission wavelength of 530 nm. Under the optimum chromatographic conditions, a linear relationship with good correlation coefficient ( r = 0.9995, n = 5) was found between the peak area ratio of RBX to PXT and RBX concentration in the range of 2–500 ng mL −1, with limits of detection and quantification of 0.5 and 1.7 ng mL −1, respectively. The intra- and inter-day precisions were satisfactory; the relative standard deviations were 2.25 and 3.01% for the intra- and inter-day precisions, respectively. The accuracy of the method proved as the mean recovery values were 100.11 ± 2.24% and 100.99 ± 2.98% for the intra- and inter-day assay runs, respectively. The proposed method involved simple and minimum sample preparation procedure and short run-time (<12 min) and therefore it can be applied to the routine therapeutic monitoring and pharmacokinetic studies of RBX.

Single hollow fiber SLM extraction of polyamines followed by tosyl chloride derivatization and HPLC determination

Determination of polyamines in biological fluids possesses medical diagnostic relevance. Despite the vast panel of analytical methods developed for polyamines they are not applied in routine clinical usage, mainly due to the time and labor consuming sample preparation step and complicated derivatization procedures. Thus, new simpler methods are needed. This paper describes a single hollow fiber SLM extraction method of polyamines followed by simple pre-column derivatization with tosyl chloride and HPLC-UV analysis. The influence of different parameters such as the extraction time, organic phase composition, acceptor pH, donor pH, acceptor volume, donor volume and stirring speed on the transport parameters and enrichment was studied and discussed. The optimized method was applied to real matrices such as urine and plasma.

Jones oxidation and high performance liquid chromatographic analysis of cholesterol in biological samples

A simple pre-column derivatization procedure for HPLC analysis of cholesterol in biological samples was developed. Cholesterol was treated with chromic acid and sulfuric acid in acetone (the Jones oxidation) and cholest-4-en-3,6-dione was formed. The reaction was finished in 5 min at room temperature and the product showed a strong UV absorbance at 250 nm that enabled an HPLC detection limit of 0.2 pmol. With stigmasterol as an internal standard, the reaction was applied to the analysis of total and free cholesterol in serum and high-density lipoproteins and the analysis showed a within-run and total coefficient of variation of about 0.2% and 0.5%, respectively.

Simultaneous determination of 5-hydroxyindoles and catecholamines by HPLC with fluorometric precolumn derivatization.

We have developed a highly sensitive and simple precolumn derivatization method for simultaneous determination of 5-hydroxyindoles (5HIs) and catecholamines (CAs) with 4-dimethylaminobenzylamine (DMBA). The method was successfully applied to the determination of 5HIs and CAs in plasma and urine.

Analysis of physiological amino acids using dabsyl derivatization and reversed-phase liquid chromatography.

A method is described for the measurement of the specific radioactivity of primary amino acids in physiological samples. The amino acids were dabsylated followed by separation using high-performance liquid chromatography. We measured the concentration of amino acids from rat plasma or liver samples. Chromatographic analyses resolved phenylalanine from a mixture of amino acids in plasma within 30 min. An extended chromatographic gradient program completely separated all physiological amino acids within 75 min. This method is as sensitive as any current method of amino acid analysis and offers several advantages including (1) simple pre-column derivatization and (2) stability of derivatized samples.