Justyna Piechocka

The article presents the first method based on high-performance liquid chromatography and ultraviolet detection (HPLC-UV) for the determination of timonacic (thioproline, 1,3-thiazolidine-4-carboxylic acid, tPro) in pharmaceutical tablets and face care products (creams, sera, foundations, suncreams). Sample preparation primarily involves solid-liquid extraction (SLE) of tPro with 0.2 mol/L phosphate buffer pH 6, derivatization with 0.25 mol/L 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT), followed by polytetrafluoroethylene (PTFE) membrane filtration. The chromatographic separation of the stable UV-absorbing 2-S-quinolinium derivative is achieved within 14 min at 25 °C on a Zorbax SB-C18 (150 × 4.6 mm, 5 µm) column using gradient elution. The eluent consists of 0.1 mol/L trichloroacetic acid (TCA), pH 1.7, in a mixture with acetonitrile (ACN) delivered at a flow rate of 1 mL/min. The analyte is quantified by monitoring at 348 nm. The assay linearity was observed within 0.5–125 μmol/L. The limit of quantification (LOQ) was found to be 0.5 μmol/L. The accuracy ranged from 93.22% to 104.31% and 97.38% to 103.48%, while precision varied from 0.30% to 11.23% and 1.13% to 9.64% for intra- and inter-assay measurements, respectively. The method was successfully applied to commercially available on the Polish market pharmaceutical and cosmetic products.

This report presents the first method for simultaneous determination of the 2-S-lepidinium derivatives of total α-lipoic acid (LA), homocysteine (Hcy), cysteinylglycine (CysGly), and cysteine (Cys) in human saliva, using capillary electrophoresis with pH-mediated sample stacking and ultraviolet detection (CE-UV) at 355 nm. Electrophoretic separation is carried out at 20 kV and 25 °C using a standard fused silica capillary (effective length 91.5 cm, inner diameter 75 µm). The background electrolyte consists of 0.5 mol/L lithium acetate buffer, adjusted to pH 3.5 with 0.5 mol/L acetic acid. The limit of quantification was determined to be 1 µmol/L for LA and 0.17 µmol/L for Hcy, 0.11 µmol/L for CysGly, and 0.10 µmol/L for Cys in saliva samples. Calibration curves demonstrated linearity over the concentration range of 3 to 30 µmol/L for all analytes. Method precision did not exceed 4.7%, and accuracy ranged from 87.9% to 114.0%. The developed method was successfully applied to saliva samples from eleven apparently healthy volunteers to determine the content of LA, Hcy, CysGly, and Cys. The Hcy, CysGly, and Cys concentrations ranged from 0.55 to 13.76 µmol/L, 0.89 to 9.29 µmol/L, and 1.73 to 12.99 µmol/L, respectively. No LA-derived peaks were detected in the native saliva samples.

The report presents first high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) based method for the determination of homocysteine thiolactone (HTL) in human urine. Sample preparation procedure chiefly involves chloroform liquid-liquid extraction of HTL and its re-extraction with hydrochloric acid, followed by evaporation to dryness and treatment of the residue with 1-benzyl-2-chloropyridinium bromide and perchloric acid. The chromatographic separation of obtained UV-absorbing 2-S-pyridinium derivative is achieved within 6 min at 25 °C on Zorbax SB-C18 (150 × 4.6 mm, 5 μm) column using gradient elution. The eluent consists of 0.1% acetic acid in a mixture with acetonitrile delivered at a flow rate of 1 mL/min. The analyte is quantified by monitoring at 316 nm. The assay linearity was observed within 0.1-1.0 µmol/L in urine. The limit of quantification was found to be 100 nmol/L. The accuracy ranged from 92.56 to 112.51% and 92.56–114.31%, while precision varied from 5.89 to 11.27% and 3.85–14.25% for intra- and inter-assay measurements, respectively. The method was successfully applied to urine samples delivered by apparently healthy donors. The presence of HTL was confirmed in twelve of eighteen study samples. The HTL concentration ranged from 36.67 to 693.12 nmol/L.

Sulfur-containing amino acids, including cysteine (Cys) and homocysteine (Hcy), as well as aldehydes such as formaldehyde (FA), acetaldehyde (AA), pyridoxal 5`-phosphate (PLP), acrolein (ACR), malondialdehyde (MDA), croton aldehyde (CA) and 4-hydoxynonenal (4-HNE) widely occur in the human body. As indicated in the literature, abnormal (elevated) concentrations of these compounds in humans may result in or even be the cause of the occurrence/development of some civilization diseases. At the same time, the literature provides the evidence that Cys and Hcy are a highly reactive towards aldehydes resulting in formation of substituted thiazolidine and thiazine carboxylic acids, respectively. Importantly, it has been shown that these adducts are formed in vitro and in vivo, while chromatographic techniques are suitable for their monitoring in biological samples. In this work, the above-mentioned issues are discussed. In particular, Cys and Hcy as well as selected endogenous aldehydes are characterized, and their role in human body in health and disease is discussed. Moreover, chromatographic methods enabling determination of selected adducts of Cys and Hcy with the above-mentioned aldehydes in biological samples are described.

The report presents first high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine and active form of vitamin B6 pyridoxal 5′-phosphate. The assay employs 4-deoxypyridoxine (4-DPD) as an internal standard. Sample preparation procedure primarily involves acetonitrile (ACN) extraction of HPPTCA from plasma proteins, sample deproteinization by ultrafiltration, and dilution of ultrafiltrate with mobile phase prior to chromatographic analysis. The chromatographic separations of HPPTCA and 4-DPD are achieved within 6 min at 20 °C on X-Bridge Glycan BEH Amide (100 × 2.1 mm, 2.5 μm) column using gradient elution. The eluent consists of 0.1% formic acid in a mixture of solvent A (water and ACN (95:5, v: v)) and solvent B (water and ACN (5:95, v: v)) delivered at a flow rate of 0.3 mL/min. The assay linearity was observed within 0.25-10 µmol/L in plasma. The limit of quantification was found to be 0.25 µmol/L. The method was successfully applied to plasma samples delivered by apparently healthy donors showing that the HPLC-MS/MS assay is suitable for human plasma screening. The presence of HPPTCA was confirmed in eleven of fifteen study samples. The HPPTCA concentration ranged from 0.55 to 8.39 µmol/L.

The report presents robust and high throughput methods, based on liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD), for the simultaneous determination of major metabolites of ibuprofen (IBU), namely 2-hydroxyibuprofen and carboxyibuprofen (method A) as well as creatinine (Crn) (method B) in human urine. The assays primarily involve straightforward sample purification. For both methods, the chromatographic separation of the analytes is achieved within 8 min at room temperature on Poroshell 120 SB-C18 (75 × 4.6 mm, 2.7 µm) column using gradient elution. The eluents consisted of 0.1% formic acid in water and acetonitrile (method A) or water and methanol (method B) delivered at a flow rate of 1 or 0.5 mL/min, respectively. In relation to metabolites of IBU, the assay linearity was observed within 0.06–0.5 g/L in urine, while the Crn assay linearity was demonstrated within 0.5–30 mmol/L in urine. The limit of quantification for IBU metabolites was determined to be 0.06 g/L, and 0.5 mmol/L for Crn. These methods were successfully applied to urine samples delivered by ten apparently healthy donors showing that the HPLC-ELSD assays are suitable for human urine screening.

The report presents the first method for simultaneous determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine (Cys) and active form of vitamin B6 pyridoxal 5′-phosphate (PLP), as well as total low molecular-weight thiols content, including Cys, homocysteine (Hcy), cysteinyl-glycine (Cys-Gly), and glutathione (GSH). The assay is based on high performance liquid chromatography coupled with ultraviolet detection (HPLC–UV) and involves disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP), derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by sample deproteinization with perchloric acid (PCA). The chromatographic separation of obtained stable UV-absorbing derivatives is achieved on ZORBAX SB-C18 (150 × 4.6 mm, 5.0 µm) column using gradient elution with eluent consisted of 0.1 mol/L trichloroacetic acid (TCA), pH 1.7 and acetonitrile (ACN), delivered at a flow rate 1 mL/min. Under these conditions, the analytes are separated within 14 min at room temperature, and quantified by monitoring at 355 nm. Regarding HPPTCA, the assay linearity was demonstrated within a 1–100 µmol/L in plasma and the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). The accuracy ranged from 92.74 to 105.57% and 95.43 to 115.73%, while precision varied from 2.48 to 6.99% and 0.84 to 6.98% for intra- and inter-day measurements, respectively. The utility of the assay was proved by application to plasma samples delivered by apparently healthy donors (n = 18) in which the HPPTCA concentration ranged from 19.2 to 65.6 µmol/L. The HPLC–UV assay provides complementary tool for routine clinical analysis, facilitating further studies on the role of aminothiols and HPPTCA in living systems.