TFA In Water Research Articles

Application of newly developed and validated LC-MS/MS method for pharmacokinetic study of adagrasib and pembrolizumab simultaneously in rat plasma

Non-small cell lung cancer (NSCLC) is a significant subtype of lung cancer, and poses a dangerous global threat. One of the current approaches of NSCLC treatment is a combination therapy of adagrasib and pembrolizumab. Accurate monitoring of these drug concentrations in biological fluids is critical for treatment efficacy. Since no method was reported for simultaneous estimation of these drugs, this study focuses on the development of a validated LC-MS/MS bioanalytical method for simultaneous quantification of Adagrasib and Pembrolizumab in rat plasma. The analytes were extracted from the biological matrix through liquid–liquid extraction techniques using acetonitrile as extraction solvent. The analytes were separated on a Waters X-bridge phenyl C18 column, with a mixture of acetonitrile: 0.1 % TFA in water (50: 50 v/v) as mobile phase at an isocratic flow rate of 1.0 mL/min with a runtime of about 5 min. Adagrasib (m/z 605.12 → 201.62), Pembrolizumab (m/z 146.32 → 85.15), and Sotorasib (m/z 561.59 → 218.92) were determined by recording the mass spectra through multiple reaction monitoring in positive mode. The method was validated according to USFDA guidelines. The results demonstrate satisfactory linearity with an r2 value of 0.9998 in the ranges of 40–800 and 10–200 ng/mL, accuracy with mean percentage recovery of 95.22–98.59 % and 96.98–98.57 %, precision indicated with %RSD ranged between 0.39–1.91 % and 0.85–9.03 % for Adagrasib and Pembrolizumab respectively, and other key parameters. The developed method can determine the pharmacokinetic parameters to indicate the efficacy and safety of the drugs, and also can quantify selected drugs simultaneously in biological samples.

An isocratic RP-HPLC-UV method for simultaneous quantification of tizanidine and lidocaine: application to in vitro release studies of a subcutaneous implant.

Implantable devices have been widely investigated to improve the treatment of multiple diseases. Even with low drug loadings, these devices can achieve effective delivery and increase patient compliance by minimizing potential side effects, consequently enhancing the quality of life of the patients. Moreover, multi-drug products are emerging in the pharmaceutical field, capable of treating more than one ailment concurrently. Therefore, a simple analytical method is essential for detecting and quantifying different analytes used in formulation development and evaluation. Here, we present, for the first time, an isocratic method for tizanidine hydrochloride (TZ) and lidocaine (LD) loaded into a subcutaneous implant, utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with a UV detector. These implants have the potential to treat muscular spasticity while providing pain relief for several days after implantation. Chromatographic separation of the two drugs was accomplished using a C18 column, with a mobile phase consisting of 0.1% TFA in water and MeOH in a 58 : 42 ratio, flowing at 0.7 ml min-1. The method exhibited specificity and robustness, providing accurate and precise results. It displayed linearity within the range of 0.79 to 100 μg ml-1, with an R2 value of 1 for the simultaneous analysis of TZ and LD. The developed method demonstrated selectivity, offering limits of detection and quantification of 0.16 and 0.49 μg ml-1 for TZ, and 0.30 and 0.93 μg ml-1 for LD, respectively. Furthermore, the solution containing both TZ and LD proved stable under various storage conditions. While this study applied the method to assess an implant device, it has broader applicability for analysing and quantifying the in vitro drug release of TZ and LD from diverse dosage forms in preclinical settings.

HPLC analysis of biomarkers of Toluene and Xylene in human urine samples

Introduction: Urinary Hippuric acid (HA) and Methylhippuric acid (MHA) are considered reliable biomarkers for monitoring exposure to toluene and xylenes. A simple and robust HPLC method for simultaneous determination of HA and MHA in urine samples was developed and validated as per Internation Conference on Harmonization (ICH) Q2 guidelines. Methods: Mobile phase consisting of two solvents A & B, solvent A was 0.1% TFA in water, and solvent B was 0.1% TFA in acetonitrile. Separation was performed in gradient elution mode and the time program was as follows - Time (min)/%B: 0/20, 4/20, 11/80, 11.1/90, 16/90, 16.1/20, 25/20. The detection wavelength was 205 nm. Results: The limits of detection (LOD) and quantification (LOQ) of HA in urine were 0.3 and 1.0 mg/L, and for MHA were 0.25 mg/L and 1.0 mg/L respectively and the recovery for HA and MHA were 95% and 98.9% respectively. Conclusion: The developed method was successfully applied for the analysis of urine samples.

Metal‐Free Hydrogenation of N‐Heterocycles with Trimethylamine Borane and TFA in Aqueous Solution

AbstractA metal‐free hydrogenation of quinoxalines and indoles with Me3N ⋅ BH3 and TFA in water at room temperature is reported. The protocol is featured with the usage of environmentally friendly water as the solvent, no need of metal catalyst and broad substrate scope. Sensitive functional groups such as OH‐, NH2‐, Cl‐, Br‐, especially ‐CN and ‐CO2Me were well tolerated. Preliminary mechanistic studies suggested that TFA and Me3N ⋅ BH3 provided the proton and hydride sources for the product, respectively.magnified image

Alternative methods to assess the impurity profile of a monographed API using acarbose as an example

Even though the impurity analysis of the Ph. Eur. is considered well-studied, its methodology should be reviewed periodically to ensure that it is working properly, and all impurities are captured by the section „Related substances” of the monograph. Within this study, the biotechnological produced antidiabetic drug acarbose was chosen to demonstrate some of the advantages as well as the shortcomings arising from the current related substances test of acarbose. Due to its weak chromophore, acarbose is studied by UV detection at 210 nm after being separated on aminopropyl-silyl stationary phases. Thus, the use of alternative detection techniques, such as charge aerosol detection (CAD) and a volatile mobile phase can be beneficial here. Since a simple method transfer to a mobile phase usable with the CAD was not possible, more stable stationary phases were tested. For the rapid determination of the sum of impurities, a method was developed using a pentafluorophenyl column and a mobile phase of 0.1% TFA in water. Maltose and maltotriose were further identified as additional impurities of the API. Furthermore, a method was developed and validated by means of an Amide-HILIC phase, that adequately separated acarbose and all of its impurities. However, the sensitivity of this method needs to be further improved. Additionally, a method was also developed and validated, taking into account the temperature stability of graphite columns. Thus, the separation was carried out at temperatures of about 90 °C to solve the problem of anomerization of acarbose and some of its impurities. In comparison with the other developed methods, the elution order was changed and has to be confirmed, but all components were separated and detected at a sufficient LOQ of 0.10%.

Synchronized analysis of bempedoic acid and ezetimibe in pure binary mixture and their combined tablets by a new stability indicating RP-UPLC method

The primary purpose of the designed study was to develop and validate a commercial, insightful, precise and simple RP-UPLC approach with greater response for the synchronized investigation of Bempedoic acid and Ezetimibein pure form and their fixed-dose combination tablet form. A method with Phenyl XBD (100 x 2.1mm, 1.7mm) column, mobile (solvent) phase of 0.1% v/v TFA in water and acetonitrile in 60:40 v/v pumped with a flow of 0.4mL/min and a wavelength of 236nm were competently separate the BPA and EZM with excellent resolution and shorter RT about 0.43min and 0.86 min for BPA and EZM respectively.The R2 for a range of concentrations stated for the Bempedoic acid (20-120µg/mL) and Ezetimibe (1-6µg/mL) were 0.999 and 0.999 correspondingly. The LOD and LOQ values of Bempedoic acid (0.41µg/mL and 1.23µg/mL) and Ezetimibe (0.02 µg/mL and 0.07µg/mL) unveiled the sensitivity of the stated method. The method has been validated in compliance with ICH standards. Identification of degradants peaks accompanyingthe intended analyte peaks with acceptable resolution is ascertaining the stbilitiy –indicating feature of the proposed method. Hence, the proposed approach has significant acceptance in the pharmaceutical sector.

Total Synthesis of Nocardiotide A by Using a Combination of Solid‐ and Solution‐Phase Methods

AbstractNocardiotide A has been successfully synthesized using a combination of solid‐ and solution‐phase synthesis. Peptides 1 and 2 of nocardiotide A, with different C and N termini, were synthesized on 2‐chlorotrityl chloride resin. A combination of N,N,N′,N′‐tetramethyl‐O‐(1H‐benzotriazol‐1‐yl)uronium hexafluorophosphate and hydroxybenzotriazole (HBTU/HOBt) was employed as coupling reagent and the release of the linear peptides from the resin was undertaken using AcOH and trifluoroethanol (TFE) reagents resulting in linear hexapeptides with protected side chains in good yields (83.4 % and 76 %, respectively). Peptide 1, which has Ala as C terminus and Trp as N terminus, was the only peptide that was successfully cyclized without epimerization under a dilute condition (1.25×10−3 M) using HBTU (3 equiv.) and 1 % N,N‐diisopropylethylamine (DIPEA) in dichloromethane. A combination of a small residue at the C terminus and a larger residue at the N terminus was found to be effective for the cyclization. The protecting group of the crude cyclic product was then deprotected by 95 % TFA in water and the crude was then purified to give white solid product with 16 % overall yield. The peptide was then characterized by HR‐ToFMS, 1H‐ and 13C‐NMR spectroscopy. In order to ensure the absence of epimerization during cyclization, the D‐analogue of nocardiotide A was synthesized using similar synthetic protocol. The NMR data of D‐analogue was found to have a larger difference compared to the synthetic and natural product, showing there is no epimerization at C‐terminus during cyclization.

Development and validation of a novel HILIC method for the quantification of low-levels of cuprizone in cuprizone-containing chow

Cuprizone is an amide compound that has been wildly used in various animal studies, such as in the investigation of remyelination in mouse model. It is important to control the amount of cuprizone dosed in animals to be consistent as different amounts may lead to different clinical observations. Cuprizone is usually administrated as a minor component (i.e., 0.3%) of a mixture with powdered or pelleted rodent chow. Its low content, combined with the complex nature of chow, represents a significant challenge for the quantification of cuprizone in the mixture. To the best of our knowledge, no method has been reported in the literature so far. In this study, a simple, selective, and sensitive hydrophilic interaction liquid chromatographic method was developed for the quantification of cuprizone in cuprizone pre-clinical formulations. The analytical method comprises a fast ultrasound assisted extraction with acetonitrile/water as a solvent followed by gradient separation using a Waters Xbridge HILIC column with 0.1% TFA in water and acetonitrile as mobile phases and UV detection at 220 nm. The specificity, linearity, accuracy, repeatability, and limit of quantitation (LOQ) of the method were established. The method was determined to be linear in the range of 10–200 μg/mL. Accuracy was assessed by spiking a chow placebo with various amounts of a cuprizone reference standard to achieve target concentration levels and the recoveries were within the acceptance criterion of 90–110% of the target concentrations. Repeatability was demonstrated at the nominal concentration of 100 µg/mL and LOQ level of 2.5 μg/mL. This method has been demonstrated to be suitable for its intended use and has been successfully applied to the quantification of low levels of cuprizone in chow formulations. It was found that the cuprizone content in chow could varied significantly between batches and the potential causes of the variability were investigated.

Controlling the quality of maca (Lepidium meyenii) dietary supplements: Development of compendial procedures for the determination of intact glucosinolates in maca root powder products

The demand and sales of dietary supplements derived from maca (Lepidium meyenii) have skyrocketed in the last decade and a variety of related nutritional and healthcare products have mushroomed into a business with market prominence. However, the lack of standard testing protocols for quality control could jeopardize the immediate benefits of these products for public health. We describe herein the development of analytical procedures for the determination of glucosinolates (GLs), the biologically active ingredients in maca. Because of the high polarity and instability caused by enzymatic hydrolysis, GLs in maca have been exclusively analyzed using desulfated GLs. This indirect analysis requires additional sample preparation steps, which is labor-intensive, and may lose the original GLs and introduce artificial compounds. Furthermore, the reported GL profiles of maca are inconsistent and incomplete, some GLs may be structurally misidentified. In this context, we focused on direct analysis of intact GLs in maca without the enzymatic desulfation. Four GLs (sinalbin, glucolepigramin, glucolimnanthin, and glucotropaeolin) were identified as the major GLs in maca root powder. An HPLC method based on ion pair chromatography was developed to determine individual and total GLs; chromatographic separations were achieved on a Luna column (C18, 4.6 × 100 mm,3 mm) using 0.1 % TFA in water and in methanol as mobile phase in a gradient elution mode. The developed procedures were validated within the calibration range of 10–500 μg/mL. Inter- and intra-day precision were shown to be lower than 3% at all concentrations levels with recovery between 100.2 % and 103.3 %. The procedures were applied to a total of 42 maca root powder products from 11 manufacturers. Sample analysis revealed a consistent correlation of glucotropaeolin: glucolimnanthin (1: 0.19) across all products with a correlation coefficient of 0.994. The correlation in combination with total GL contents for each product could be used for authentication and GL content determination. Incorporation of the developed procedures into USP monographs will strengthen the public standards for maca powder dietary supplements.

The development and validation of a high performance liquid chromatography method to determine the radiochemical purity of [177Lu]Lu-HA-DOTA-TATE in pharmaceutical preparations

Lutetium-177 [177Lu] tetra-azacyclododecanetetra-acetic acid [DOTA]-(Tyr3)-octreotate [TATE] ([177Lu]Lu-DOTA-TATE) is a radiopeptide used for peptide receptor radionuclide therapy in patients with neuroendocrine tumours (NETs). This radiopeptide is made by labelling the ligand octreotate with Lutetium-177 using the linker DOTA. After labelling, and before clinical application quality control of the radiopeptide is needed and the radiochemical purity is assessed. Acceptance limits for radiochemical purity should be within 90–110% of the label claim for radiopharmaceuticals for diagnostic use and within 95–105% of the label claim for radiopharmaceuticals for therapeutic use. Moreover, the amount of unlabelled [177Lu]LuCl3 cannot exceed 2% of the radioactive dose. Since no monograph is available for [177Lu]Lu-DOTA-TATE in the European Pharmacopeia (Ph Eur), this article describes the development and validation of a high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection and radiodetection. A Waters Acquity Arc UHPLC system equipped with a Waters 2998 photodiode array (PDA) detector was used coupled to a Berthold Lb 514 Flowstar detector equipped with a BGO-X gamma measuring cell. A reversed phase Symmetry Shield C18 column (4.6 mm × 250 mm, 5 µm) was used for chromatographic separation. A flow of 1.5 mL/min was maintained during analysis, using 0.1% TFA in water as mobile phase A and 0.1% TFA in ACN as mobile phase B. The retention time was around 1.7 min and 13.5 min for [177Lu]LuCl3 and [177Lu]Lu-HA-DOTA-TATE, respectively. Stock solutions of [177Lu]LuCl3 were made by serial dilution and were injected to test for linearity, accuracy and precision, carry over and signal-to-noise ratio. A [177Lu]Lu-HA-DOTA-TATE sample was prepared and injected to determine the carry over. The results showed that the method is linear over a range of 0.300–130 MBq/mL, which covers the range for clinical samples, provided that the clinical sample is diluted ten times before analysis. The LLOQ can be measured accurately even after dilution, with a signal-to-noise ratio of at least 5. In short, the method is accurate, precise and sensitive and can be implemented as part of the quality control of [177Lu]Lu-HA-DOTA-TATE.

Synthesis of Tetrapeptides and Screening of their Antioxidant Properties

Background: Tetrapeptide Pro-Ala-Gly-Tyr (PAGY) is an antioxidant peptide that was isolated by Nikoo et al. (2014) from hydrolysate of skin gelatin of amur sturgeon fish (Acipenser schrenckii). This research aims to synthesize PAGY and its analogues by a solid-phase method, and to screen their antioxidant activities. Methods: PAGY and its analogues, namely, Pro-Ser-Gly-Tyr (PSGY), Pro-Ala-Phe-Tyr (PAFY), Pro- Phe-Phe-Tyr (PFFY) and Pro-Ala-Ile-Tyr (PAIY), were synthesized via a solid phase peptide synthesis method with the Fmoc/t-Bu strategy. The synthesis was undertaken on 2-chlorotritylchloride resin as solid support, and all coupling reactions were facilitated by a combination of HBTU and HOBt reagents. All peptides were cleaved from the resin by using 95% TFA in water. Results: Through the solid-phase synthesis method, all peptides were obtained in 50-85% yields. Pure peptides were analysed by analytical RP-HPLC, and were characterized by HR-TOF-ESI-MS and 1HNMR. DPPH inhibition assay was applied to all of the peptides with IC50 of 1.750; 1.116; 1.177 ;1.437; and 1.642 mg/mL for PAGY, PSGY, PAFY, PFFY, and PAIY, respectively. Conclusion: PAGY and its four analogs were successfully synthesized by solid-phase method. PSGY compound was found to have the highest activity of all in DPPH assay.

Development and Validation of a New HPLC Method for Quantification of a Novel Antifungal Drug Based on 1,3,4-Thiadiazole and its Impurities

Chromatographic behavior of a new antifungal drug, (2-[(Z)-1-(3,5-diphenyl-1,3,4-thiadiazol-2(3H)-ylidene)-methyl]-3,5-diphenyl-1,3,4-thiadiazol-3-ium chloride (TDZ), and its impurities was studied using several types of columns, mobile phases and elution modes. A new reversed phase high-performance liquid chromatography (HPLC) method for the determination of TDZ and its impurities in a pharmaceutical substance and dosage form (tablets, 60 mg) was developed and validated. The optimal chromatographic separation of TDZ and its impurities was performed on a 5-μm C8 column (4.6 mm × 250 mm, I.D.). The mobile phase consisted of 0.03% TFA in water (pH 2.5) and acetonitrile under gradient elution condition (from 40:80 v/v to 80:20 v/v for 20 min) at a flow rate of 1.0 mL min−1. UV detection was set at a wavelength of 250 nm. The limit of detection (LOD) and quantification (LOQ) were 0.05 and 0.1 μg mL−1, respectively. The method showed good linearity (r2 > 0.999) over the assayed concentration range (0.1–1.4 μg mL−1 for quantification of impurities and 0.005–0.013 mg mL−1 for quantification TDZ) and demonstrated good intra- and inter-day precision and accuracy. The method was validated and successfully applied for quantitative determination of TDZ and its impurities in both the pharmaceutical substance and tablets.

Synthesis of Antioxidant Peptide SCAP1 (Leu-Ala-Asn-Ala-Lys)

SCAP1 is a pentapeptide isolated from the hydrolysate of oyster (Saccostrea cucullata), which has antioxidant and anticancer properties. This study aims to find a novel strategy to obtain antioxidant peptide SCAP1 (Leu-Ala-Asn-Ala-Lys) through chemical synthesis. This study reports the first synthesis of SCAP1 through the solid phase peptide synthesis (SPPS) method with Fmoc strategy on 2-chlorotrityl chloride resin. A HATU-HOAt coupling reagent was employed in all amide bond formations and 95% TFA in water was employed to release the desired peptide from the solid support. The crude peptide was purified using successive preparative-RP-MPLC and RP-HPLC to obtain the pure peptide. The synthesized peptide was confirmed by mass spectrometry, showing a correct molecular ion peak at m/z 516.3143 (M+H)+, referring to the desired SCAP1. 1H-NMR spectra of the peptide revealed protons signals consistent to the structure of SCAP1. During the synthesis, the coupling between Ala and Asn(Trt) was not straightforward, which led to a low yield of the final product. The use of Trt-based Asn was not a suitable option, as it could cause aggregation during the synthesis, eventually leading to a poor yield of the final product. SCAP1 was obtained as a white solid with a percent yield of 8.28%.

Determination of residual poly diallyldimethylammonium chloride (pDADMAC) in monoclonal antibody formulations by size exclusion chromatography and evaporative light scattering detector

The cationic polyelectrolyte pDADMAC is widely used in biopharmaceutical industry as a flocculating agent to enhance clarification throughput and downstream filtration operations. Due to the possible toxicity, pDADMAC should be assessed for an acceptable residual level to ascertain the safety of the product to patients. The strong protein-polyelectrolyte interaction, however, can negatively affect sensitivity and accuracy of measurements. This paper reports on the application of size exclusion (SE) chromatography coupled to evaporative light scattering detector (ELSD) to the quantitative determination of pDADMAC in monoclonal antibody formulations and in process intermediates during downstream purification. The SE chromatography was performed under isocratic condition with a mobile phase consisting of 0.1% TFA in water (90%) and acetonitrile (10%) at a flow rate of 0.4 ml/min. A quantification limit (S/N = 10) of 0.85 ppm was achieved in sample matrix, which is sufficiently low for the trace analysis of this compound in protein-containing samples.

A Novel Ultra-High Performance Liquid Chromatography Method for the Determination of Erdosteine, Related Impurities and Degradation Products in New Effervescent Tablets

A novel and automated, stability-indicating, reversed phase ultra performance liquid chromatography (UPLC) method was developed and validated for the quantitative determination of erdosteine, its known impurities and two novel degradation products in a new pharmaceutical dosage form (effervescent tablets). The chromatographic separations were performed on a Waters Acquity UPLC HSS T3, 1.8 µm (2.1 mm × 150 mm, I.D.) stainless steel column. The mobile phase consisted of 0.1% TFA in water and methanol under gradient elution conditions, at a flow rate of 0.29 mL/min, for the assay and impurities analysis. UV detection was set at a wavelength of 238 nm. Erdosteine raw material, placebo and effervescent tablets were subjected to forced degradation. The new degradation products (labeled OX1 and OX2) were found after oxidative treatment and characterized by ultra performance liquid chromatography mass spectrometry. The validation parameters such as linearity, limit of detection (LOD) and quantification (LOQ), accuracy, precision, specificity and robustness were highly satisfactory for all analyzed compounds. LOD (0.020 and 0.011–0.385 µg/mL for erdosteine and impurities, respectively) and LOQ values show the high sensibility of the method. Specificity of the method was confirmed by testing the matrix components. The validated method demonstrated to be suitable for routine quality control purposes and for routine stability studies of erdosteine in effervescent formulations.

Solid-phase Synthesis of Tetrapeptide on 2-Chlorotrityl Chloride Resin by Using Benzotriazol-1-yl-oxytripyrrolidinophosphonium Hexafluorophosphate as Coupling Reagent

Tetrapeptide, OH-Pro-Ala-Gly-Tyr-NH 2 , was successfully synthesised on 2-chlorotrityl chloride resin by taking advantage of PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) as coupling reagent. The selection of the peptide as the target of synthesis was due to its interesting bioactivity as antioxidant. The synthesis was undertaken with Fmoc strategy, where Fmoc-proline was added onto the resin at the first place. It is known from the literature that proline can resist from rasemisation when it was attached on the resin at the first time. Fmoc deprotection step was carried out by employing 20% piperidine in DMF and the reaction mixture was shaken for 30 minutes. Once the proline attached, the next step was to attach amino acids, alanine (Ala), glycine (Gly) and tyrosine(Tyr), subsequently onto the resin until tetrapeptidyl resin was constructed on the resin. Hydroxyl group of Tyr was protected with t-butyl, which is TFA-labiled. Coupling reaction was undertaken by mixing the amino acid and PyBOP in a mixture of dichloromethane and DMF (1:1) and in the presence of basic DIPEA. Resin cleavage step was carried out by using 95% TFA in water, where t-butyl protecting group on the side chain of Tyr was cleaved at the same time. The analytical RP-HPLC of the final product showed a single peak at 21.9 minutes (20-90% of acetonitrile in water with 0.1% of TFA during 30 minutes), indicating that each coupling has given a good coupling performance and resulting in a pure product. The desired product showed the correct molecular weight with m/z 407.2 [M+H] + and 429.2 [M+Na]. DOI: http://dx.doi.org/10.15408/jkv.v0i0.4055

Solid-phase Synthesis of Tetrapeptide on 2-Chlorotrityl Chloride Resin by Using Benzotriazol-1-yl-oxytripyrrolidinophosphonium Hexafluorophosphate as Coupling Reagent

Tetrapeptide, OH-Pro-Ala-Gly-Tyr-NH2, was successfully synthesised on 2-chlorotrityl chloride resin by taking advantage of PyBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) as coupling reagent. The selection of the peptide as the target of synthesis was due to its interesting bioactivity as antioxidant. The synthesis was undertaken with Fmoc strategy, where Fmoc-proline was added onto the resin at the first place. It is known from the literature that proline can resist from rasemisation when it was attached on the resin at the first time. Fmoc deprotection step was carried out by employing 20% piperidine in DMF and the reaction mixture was shaken for 30 minutes. Once the proline attached, the next step was to attach amino acids, alanine (Ala), glycine (Gly) and tyrosine(Tyr), subsequently onto the resin until tetrapeptidyl resin was constructed on the resin. Hydroxyl group of Tyr was protected with t-butyl, which is TFA-labiled. Coupling reaction was undertaken by mixing the amino acid and PyBOP in a mixture of dichloromethane and DMF (1:1) and in the presence of basic DIPEA. Resin cleavage step was carried out by using 95% TFA in water, where t-butyl protecting group on the side chain of Tyr was cleaved at the same time. The analytical RP-HPLC of the final product showed a single peak at 21.9 minutes (20-90% of acetonitrile in water with 0.1% of TFA during 30 minutes), indicating that each coupling has given a good coupling performance and resulting in a pure product. The desired product showed the correct molecular weight with m/z 407.2 [M+H]+ and 429.2 [M+Na]. DOI: http://dx.doi.org/10.15408/jkv.v0i0.4055

Substituent effects on the kinetics of acid-catalyzed hydrolysis of methyl cellulose

The kinetics of the hydrolysis of methyl cellulose (MC, DS 1.27 and 1.95) was studied by a two-step procedure, comprising partial hydrolysis in 1 M TFA in water and water/acetone at 120 °C for various time periods, labeling of generated reducing ends by reductive amination, complete depolymerization by methanolysis followed by trimethylsilylation, and gas chromatographic analysis of the two sets of partially O-methylated glucose derivatives. Rate constants of MCs were all in the order of 10−4 s−1. In aqueous TFA, overall rate of hydrolysis of the MC with lower DS was faster than of the MC with higher DS. When substituting half of the water by acetone, reaction was slowed down while selectivity regarding different O-methyl glucosyl residues increased. Compared to the parent glucosyl unit methylation at O-2 and at O-6 decreased rate of hydrolysis, while 3-O-methyl favored it especially in the early stage of the conversion of the macromolecules. Beside slight differences between the two MCs and reaction conditions, rate constants k i (i = position of methyl) followed the order k 36 ≈ k 3 > k 0 ≈ k 23 > k 6 > k 2 ≥ k 236 > k 26. For the higher substituted MC2 an initial slow phase with more pronounced differences of k i, followed by a faster less selective period was observed. Regioselectivity of hydrolysis with respect to methyl positions was expressed as standard deviation of k i and was between 16 and 46% depending on MC and conditions. Findings are discussed with respect to electronic effects, solvent-effect, H-bonding pattern and solution state.

Comparison of different sorbent materials for solid-phase extraction of selected drugs in human urine analyzed by UHPLC–UV

A procedure based on solid-phase extraction (SPE) followed by ultra-high-performance liquid chromatography (UHPLC) with UV detection has been developed for the analysis of multiple drugs in human urine. The compounds evaluated were aliskiren, prasugrel, rivaroxaban, prednisolone, propranolol, ketoprofen, nifedipine, naproxen, terbinafine, ibuprofen, diclofenac, sildenafil and acenocoumarol. Seventeen different solid phase extraction (SPE) cartridges were tested to evaluate their applicability for the isolation of drugs from human urine. Comparison were recovery of different drugs and reproducibility. The samples were analyzed by UHPLC using a Poroshell 120 EC-C18 column and acetonitrile -0.05% TFA in water as the mobile phase under gradient elution conditions. SPE combined with UHPLC-UV allowed the determination of drugs over a linear range of 0.01-30.0μg/mL, with limits of detection at 0.003-0.217μg/mL and precision of 0.8-7.1%. Phenyl (C6H5) sorbent was found to provide the most effective clean-up, removing the greatest amount of interfering substance and simultaneously ensuring analyte recoveries higher than 85.5% with relative standard deviations (RSD) <10%. The method was applied with good accuracy and precision in the determination of drugs in human urine obtained from patients treated with selected drugs.

Multiresidue LC–MS/MS analysis of cephalosporins and quinolones in milk following ultrasound‐assisted matrix solid‐phase dispersive extraction combined with the quick, easy, cheap, effective, rugged, and safe methodology

A sensitive and selective confirmatory method for milk-residue analysis of ten quinolones and eight cephalosporins by LC-MS/MS has been developed herein. For the chromatographic separation of target analytes, a Perfectsil ODS-2 (250 × 4 mm, 5 μm) analytical column was used and gradient elution was applied, using a mobile phase of 0.1% w/w TFA in water and 0.1% w/w TFA in ACN. Ultrasound-assisted matrix solid-phase dispersion procedure was applied for the extraction and clean-up procedure of antimicrobials agents from milk matrix using a mixture of Bond Elut Plexa sorbent and QuEChERS. The method was validated meeting the European Legislation determining selectivity, linearity response, trueness, precision (repeatability and between-day reproducibility), decision limit, detection capability, and ruggedness following the Youden approach. Recoveries of all antibiotics ranged from 81.7 to 117.9%, while RSD values were lower than 13.7%. Limits of quantification for all examined compounds ranged from 2.4 to 15.0 μg/kg, substantially lower than the maximum residue limits established by the European Union (30-100 μg/kg).