C18 Column Research Articles

A novel HPLC method for selexipag in human plasma and application to a prototype pharmacokinetic study

AbstractA novel simple and cost effective HPLC technique was presented for the quantification of selexipag (SLP) in human plasma sample and the technique's applicability to a pharmacokinetic investigation. Chromatographic separation was achieved with C18 (5 µm × 4.6 mm × 150 mm) column, at 30 °C with isocratic elution, mobile phase composed of solution A (acetonitrile), and solution B (0.5% formic acid) (65:35 v/v) at flow rate 1.2 mL min−1. The linearity range is 10–150 ng mL−1. As sample preparation step human plasma was precipitated with acetonitrile and the detection was provided at 300 nm. The retention time is 8.20 ± 0.02 min. LOD is found to be 3.3 ng mL−1 for drug. The method was applied to the analysis of SLP in human plasma with good recovery as 97.83%. Validation of the studied methods was carried out according to EMA guideline. The new method applied on a prototype pharmacokinetic study by administration of 800 μg SLP to a healthy volunteer and parameters like AUC0–24, AUC0–∞, Cmax, tmax, and t1/2 were assessed.

Determination of eight neonicotinoid pesticides in wastewater by solid phase extraction combined with liquid chromatography-tandem mass spectrometry

Neonicotinoid pesticides are a relatively new class of pesticides that have garnered significant attention owing to their potential ecological risks to nontarget organisms. A method combining solid phase extraction with liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed for the rapid and accurate detection of eight neonicotinoid pesticides (dinotefuran, E-nitenpyram, thiamethoxam, clothianidin, imidacloprid, imidaclothiz, acetamiprid, and thiacloprid) in wastewater. The chromatographic mobile phase and MS parameters were selected, and a single-factor method was used to determine the optimal column type, extraction volume, sample loading speed, and pH for SPE. The optimal parameters were as follows: column type, HLB column (500 mg/6 mL); sample extraction volume, 500 mL; sample loading speed, 10 mL/min; and sample pH, 6-8. The matrix effects of the wastewater samples were reduced by optimizing the chromatographic gradient-elution program, examining the dilution factor of the samples, and using the isotope internal standard calibration method. Prior to analysis, the wastewater samples were diluted 5-fold with ultrapure water for pretreatment. Subsequently, 2 mmol/L ammonium acetate aqueous solution containing 0.1% (v/v) formic acid and methanol was used as mobile phases for gradient elution on a ZORBAX Eclipse Plus C18 column (100 mm×2.1 mm, 1.8 μm). The samples were quantified using positive-ion multiple reaction monitoring (MRM) mode for 10 min. Imidacloprid-d4 was used as the isotope internal standard. The SPE process was further optimized by applying response surface methodology to select the type and mass of rinsing and elution solvents. The optimal pretreatment of the SPE column included rinsing with 10% methanol aqueous solution and elution with methanol-acetonitrile (1∶1, v/v) mixture (7 mL). The eight neonicotinoid pesticides showed satisfactory linearity within the relevant range, with linear correlation coefficients (r) all greater than 0.9990. The method detection limits (MDLs) ranged from 0.2 to 1.2 ng/L, and the method quantification limits (MQLs) ranged from 0.8 to 4.8 ng/L. The average recoveries of the eight neonicotinoid pesticides were in the range of 82.6%-94.2% at three spiked levels, with relative standard deviations (RSDs) ranging from 3.9% to 9.4%. Finally, the optimized method was successfully applied to analyze wastewater samples collected from four sewage treatment plants. The results indicated that the eight neonicotinoid pesticides could be generally detected at concentrations ranging from not detected (ND) to 256 ng/L. The developed method has a low MDL and high accuracy, rendering it a suitable choice for the trace detection of the eight neonicotinoid pesticides in wastewater when compared with other similar methods. The proposed method can be utilized to monitor the environmental impact and assess the potential risks of neonicotinoid pesticides in wastewater, thus promoting the protection of nontarget organisms and the sustainable use of these pesticides in agriculture.

Simultaneous determination of 61 hormones in water by solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry

Concerns over the emergence of steroid hormones as pollutants in water have grown. Steroid hormone compounds present challenges in the simultaneous detection of total residual hormones owing to their analogous structures and diverse types. In this study, we established a rapid and high-throughput continuous online method based on solid phase extraction (SPE) coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for the simultaneous determination of 61 hormone components, including 48 glucocorticoids, 1 mineralocorticoid, 4 androgens, and 8 progesterones, in water. Various SPE columns were investigated to assess their extraction efficiency for enriching and purifying target compounds in a large sample volume (1 L). An HC-C18 SPE column was selected because of its superior performance. Acetonitrile was used as a washing solution during SPE to ensure that the majority of the tested substances achieved recoveries exceeding 70% and effectively avoid interferences from water-soluble components. Various C8 and C18 columns were tested, and the optimal HPLC conditions for hormone retention were established. We systematically evaluated different UPLC columns and mobile phases, including methanol-water and acetonitrile-water systems with 0.1% formic acid added to the aqueous phase. The optimized UPLC conditions were as follows: BEH C18 column (100 mm×2.1 mm, 1.7 μm); column temperature, 40 ℃; flow rate, 0.3 mL/min; injection volume, 5 μL; mobile phase A: 0.1% formic acid aqueous phase; mobile phase B: acetonitrile. Gradient elution was performed as follows: 0-0.5 min, 30%B; 0.5-15.0 min, 30%B-75%B; 15.0-18.0 min, 75%B-98%B; 18.0-19.0 min, 98%B; 19.0-19.1 min, 98%B-30%B; 19.1-20.0 min, 30%B. Both positive- and negative-ion modes were explored in the UPLC-MS/MS experiment to obtain the full scan of the parent ions, and positive mode was finally selected for electrospray ionization (ESI). Two product ions exhibiting strong signals and minimal interference were selected for quantitative and qualitative ion analyses, using an external standard method for quantification. MS/MS was performed in positive-ion (ESI+) mode with multiple reaction monitoring (MRM) scanning. The MS/MS parameters were as follows: atomizing gas pressure, 379 kPa; curtain air pressure, 241 kPa; spray voltage, 5500 V; desolvation temperature, 550 ℃; collision exit voltage (CXP), 13 V; intake voltage (EP), 10 V; and residence time of each ion pair, 0.5 ms. Other instrument settings, such as the collision energy and declustering voltage, were also optimized. The 61 hormones exhibited excellent linear relationships within their corresponding concentration ranges, with correlation coefficients greater than 0.99. The method detection limits (MDLs) were in the range of 0.05-1.50 ng/L. The average recoveries of the 61 hormones across three spiked levels ranged from 62.3% to 125.2%, with relative standard deviations (RSDs, n=6) of 1.1%-10.5%. Using this method, we successfully detected 10 hormone components (cortisone, fluticasone propionate, ciclesonide, betamethasone dipropionate, clobetasone butyrate, diflucortolone valerate, halobetasol propionate, isoflupredone, difluprednate, and hydroxyprogesterone caproate) in various surface water and groundwater samples collected from the Taihu Basin region. The SPE-UPLC-MS/MS method presented in this paper is simple, highly sensitivity, and exceptionally accurate. Thus, it exhibits promising potential for tracing targeted hormone residues in water and will be of great value in monitoring and ensuring water safety. Finally, a regional analysis was conducted on the hormone levels in water, and suggestions were made for the targeted treatment of hormone residues in future sewage treatment processes.

Identification of chemical components of large-leaf yellow tea by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry

Large-leaf yellow tea, a slightly fermented yellow tea that is unique to China, has a stronger hypoglycemic effect than other tea varieties, such as green and black tea. Research on large-leaf yellow tea has focused on its hypoglycemic effect owing to the lack of comprehensive techniques to characterize its chemical components; thus, its development and further promotion are limited. Therefore, the development of a reliable analytical method to fully characterize the chemical components of large-leaf yellow tea is urgently required. In this study, a reliable strategy based on the data-acquisition technology of ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q TOF/MS) was established to rapidly screen and analyze the main chemical components of large-leaf yellow tea by combining the information of neutral loss groups and characteristic fragment ions. The chromatographic separation experiments were performed on a Waters ACQUITY UPLC BEH C18 column (100 mm×2.1 mm, 1.7 μm) with gradient elution using 0.1% formic acid aqueous solution and acetonitrile as the mobile phases. The flow rate was 0.2 mL/min, the sample volume was 2 μL, and the column temperature was 35 ℃. The mass spectral information of the components in a large-leaf yellow tea solution was collected using the full-information tandem MS (MSE) technique in positive and negative ion modes. The specific chemical components of large-leaf yellow tea was identified as follows. First, a self-established database of tea chemical components was constructed based on the literature. The mass spectral cleavage pathways of different types of compounds in large-leaf yellow tea were then sorted using reference substances, and the characteristics of the fragment ions and neutral loss groups were summarized. The precise mass-to-charge ratio of the target chemical components were then obtained based on the mass spectral information. Finally, the structures of the compounds in large-leaf yellow tea were confirmed based on their chromatographic retention times, mass spectral cleavage pathways, characteristic fragment ions, and neutral loss groups. A total of 87 chemical components, including 10 catechins, 32 flavonoids, 16 phenolic acids, 12 tannins, 6 theaflavins, and 11 compounds in other classes, were identified in large-leaf yellow tea. Representative compounds of various classes, including gallocatechin gallate, quercetin, vitexin, gallic acid, chlorogenic acid, 1,3,6-tri-O-galloyl-β-D-glucose, and theaflavin, were selected, and their characteristic fragment ions and neutral loss groups were investigated in detail to reveal the cleavage pathways of different types of compounds in large-leaf yellow tea. The UPLC-Q TOF/MS method established in this study can comprehensively identify the main chemical components of large-leaf yellow tea in a simple, highly sensitive, stable, and reliable manner. This study provides a scientific basis and data support for the discovery of functional ingredients and quality evaluation of large-leaf yellow tea.

Efficacy of the peak area method of high-performance liquid chromatography (HPLC) analysis: The separation, identification, and quantification of phytochemical compounds of Moringa oleifera

The quantification and identification of phytochemical compounds in Moringa oleifera have received significant attention due to their potential health benefits. High-Performance Liquid Chromatography (HPLC) has become a widely used method for analyzing these phytochemicals due to its speed, adaptability, and effectiveness. However, there is a need to improve these methods to achieve more accurate quantification and understanding of these compounds. This study aims to assess the effectiveness of the peak area method in HPLC for the separation, identification, and quantification of phytochemical compounds in Moringa oleifera leaves. The main goal of the study is to use the peak area method within HPLC to measure the concentration of various phytochemical compounds in Moringa oleifera leaves. This method offers a more reliable measurement than peak height, allowing for comparison of different components' concentrations within or between samples, which is important for trace analysis in various applications. The methodology involved collecting and preparing Moringa oleifera leaves from five regions: Nigeria, Ghana, Haiti, India, and Texas, USA. The leaves were dried, ground, and extracted using methanol and ethanol. The extracts were then analyzed using HPLC, with the peak area method utilized to quantify the phytochemical compounds. The HPLC system utilized a Nucleosyl C18 column with a UV-vis diode-array detector, and the separation process was monitored at 332 nm. Key findings of the study indicated that chlorogenic acid, rutin, ferulic acid, luteolin, apigenin, and kaempferol were successfully separated, identified, and quantified from Moringa oleifera leaves using the peak area method in HPLC. Chlorogenic acid had a retention time of 15.759 minutes and a peak area of 170,844, indicating it is more polar and present in significant quantities. Rutin followed with a retention time of 16.919 minutes and a peak area of 115,599. Ferulic acid was the most abundant compound, with a peak area of 422,129 and a retention time of 17.965 minutes. Luteolin, with a peak area of 235,655 and a retention time of 19.777 minutes, was also present in considerable amounts. Apigenin and kaempferol showed retention times of 20.840 minutes and 21.039 minutes with peak areas of 343,378 and 197,585, respectively. The study also compared the extraction efficiencies of 70% ethanol and 80% methanol, finding that methanol was significantly more effective, especially for highly polar compounds like chlorogenic acid, luteolin, and rutin, with peak areas being 74% to over 1100% higher than those observed with ethanol. The study concluded that the peak area method in HPLC is a highly effective tool for the separation, identification, and quantification of phytochemical compounds in Moringa oleifera leaves. It recommended further optimization of the HPLC method and exploration of pharmacological properties of these phytochemical compounds for nutraceuticals and pharmaceuticals.

Development and Validation of an ICH-Compliant Optimized RP-HPLC Method for Quantitative Analysis of Favipiravir

Favipiravir (FAV) has emerged as a promising antiviral agent. It is particularly effective against influenza and other RNA virus infections. The aim and objective of the present study was developing and optimizing chromatographic conditions for faster analysis of FAV and demonstrating its applicability for tablet assay. The chromatographic separation was obtained using mobile phase with 90:10 % v/v ratio of ammonium acetate buffer (pH 4.5) and methanol on a Waters C18 column (250 x 4.6 mm, 5 µ). A flow rate of 1.4 mL/min was optimized. Chromatographic detection was perform at a wavelength of 323 nm. The FAV retention time was 6.46 min, indicating the efficiency and speed of the method. The developed method was rigorously validated according to ICH guidelines. A strong linear correlation (r2 = 0.9995) was established in the 5–60 μg/mL range, indicating adequacy for quantitative analysis. The method demonstrated high accuracy, with FAV recovery ranging from 98.77 % to 100.89 %. The % RSD results of less than 2% for intermediate precision and repeatability showed that the method exhibited high precision. The developed HPLC method reduces the Rt. Notably, this method complies with regulatory standards, establishing it as a valuable tool for quality assurance, pharmacological evaluation, and clinical monitoring of FAV. Thus, this validated RP-HPLC method provides a robust and sensitive method for routine FAV quantification, with high accuracy, precision, and compliance results, supporting its potential use in the clinical pharmaceutical industry.

Stability Indicating Method Development and Validation for the Simultaneous Estimation of N-Acetylcysteine and Acebrophylline in Tablet Dosage form by RP-HPLC

A quick, accurate, precise Stability indicating method for Simultaneous estimation of N-Acetylcysteine and Acebrophylline in Tablet dosage form was developed and validated. Chromatogram was run on Waters C18 (150 x 4.6 mm, 2 μm) Column. Mobile phase containing OPA Buffer and Methanol taken in a ratio of 10:90 % v/v was pumped at a flow rate of 1.5 mL/min through column. Cushion utilized in this strategy was OPA. Column temperature was kept at 35°C. Injection volume was 5 μL and Run time was 5 minutes. Sample was scanned at 285 nm. RT’s of N-Acetylcysteine and Acebrophylline were found to be 2.646 min and 2.117 min respectively. The developed method was validated and was found to be Accurate, Precise and Linear over the Concentration Range of 25 % to 175 % of Test concentration. Retention time and Runtime are less and the method was properly validated so this method can be utilized for routine analysis and stability studies of Assay of N-Acetylcysteine and Acebrophylline in Tablet dosage form in industries.

MonoTip C18 pipette tip solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry enables rapid and automated therapeutic drug monitoring of tyrosine kinase inhibitors

This work presents an advanced automated monolithic C18 pipette-tip solid-phase extraction (PT-SPE) method seamlessly integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for precise therapeutic drug monitoring of eleven tyrosine kinase inhibitors (TKIs) in biological samples. Commercially available MonoTip C18 columns facilitate superior extraction efficiency and selectivity from complex biological matrices owing to their large surface areas and enhanced mass transfer capabilities. The sample preparation process, conducted by aspirating and dispensing solutions using a pipettor, completes the overall extraction process within 3 min. The method validation demonstrated excellent linearity within the range of 0.2/0.5/0.8 to 200 ng/mL, with detection limits ranging from 0.049 to 0.206 ng/mL. The method exhibited outstanding accuracy and precision, with intraday relative recovery rates ranging from 96.3 % to 115.0 % and interday recovery rates ranging from 95.0 % to 115.7 %, with coefficient of variation values consistently below 13.8 %. The proposed method is distinguished by its exceptional efficiency, reduced solvent use, enhanced environmental sustainability, and streamlined automation—key advantages in the realm of clinical therapeutic drug monitoring of TKIs. Overall, the MonoTip C18 PT-SPE technique, coupled with LC-MS/MS, offers a formidable, eco-friendly, and effective strategy for the TDM of TKIs, marking a significant advancement in the field.

An adhesive hydrogel functionalized silica sphere for polar analytes separation and analysis

In response to the challenges associated with the chromatographic separation of polar compounds, this study aims to devise a solution by introducing a novel stationary phase. Hydrogels, characterized by a three-dimensional network structure, have aroused wide attention owing to its functional designability, multiple interaction sites and good adhesion, etc. In this work, an adhesive hydrogel functionalized silica stationary phase (Sil@PVA/TA) was synthesized using physical coating technique. Due to the co-existence of hydroxyl and benzene ring in the hydrogel structure, the obtained composites materials exhibited excellent separation performance for various of compounds and excellent column efficiency up to 71385.6 plates/m for thymidine. Furthermore, the hydrogel functionalized silica demonstrated superior selectivity to bare silica, diol-column and NH2-column for the separation of various of polar molecules, including, nucleosides/bases, alkaloids, organic acids, antibiotics and amino acids. Notably, for alkaloids, which frequently encounter peak tailing issues, Sil@PVA/TA demonstrated superior peak shape compared with C18 column. In short, this study successfully synthesized a hydrogel functionalized silica stationary phase, offering a novel method for the separation and analysis of polar compounds.

Simultaneous determination of free DL-amino acids in human hair with a novel DBD-M-Pro derivatization by UHPLC-HRMS: An application in diabetes patients

Human hair is a non-invasive biological sample that is easy to collect and store and can reflect long-term body health. However, the correlation between DL-amino acids and metabolic diseases in hair samples has not been studied. Therefore, we propose a novel UHPLC-HRMS method for analyzing seven free chiral amino acids (DL-Thr, DL-Glu, DL-Ala, DL-Val, DL-Pro, DL-Leu, and DL-Phe) simultaneously in hair samples by derivatization of chiral probe 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-trans-2-methyl-L-proline (DBD-M-Pro) labeled with targeted amino functional groups. Gradient elution was carried out using an ACQUITYTM BEH C18 (100×2.1 mm,1.7 μm) column with a mobile phase of 0.15 % formic acid (FA) in 10 mM ammonium acetate (CH3-COONH4) and 0.2 % FA in acetonitrile. The labelled DL-amino acid diastereoisomers could be completely separated, with a resolution (Rs) of 1.59–11.44. These amino acids show a strong linear correlation within the range of 3.1–99.2 pmol (R2 ≥ 0.9990). Intraday and interday precision was 1.87 %–14.87 %. The average recovery was 96.12 %–105.33 %. The limit of detection (LOD) ranged from 0.29 to 2.11 pmol. We then employed the method to determine the concentration of free chiral amino acids in hair samples from 30 healthy volunteers (HVs) and 30 diabetes patients (DPs). Male diabetes patients had significantly higher levels of L-Thr, L-Val, L-Leu (p < 0.05), and D-Ala (p < 0.01) in their hair samples than male healthy volunteers and female diabetes patients had significantly higher levels of D-Ala (p < 0.05) in their hair samples than female healthy volunteers. This is the first study to confirm the feasibility of using free DL-amino acids in human hair as potential biomarkers for diabetes.

Simultaneous quantification of 2-pyrrolidone-5-carboxylic acid (PCA), urocanic acid (UCA), and histidine (His) in the stratum corneum by HPLC-PDA

Located in the epidermis, the stratum corneum is the most superficial layer of the skin, acting as a barrier against aggression from the external environment, preventing dehydration, and maintaining the water balance of the skin. The stratum corneum contains the Natural Hydration Factor (NMF), a mixture of hygroscopic molecules derived from filaggrin. The NMF includes 2-pyrrolidone-5-carboxylic acid (PCA), urocanic acid (UCA), and histidine (His), target biomarkers that were extracted by tape-stripping from the stratum corneum of participants for quantification in HPLC-PDA. By extracting the stratum corneum, we developed a protocol to optimize, through audience definition, the quantification of NMF biomarkers. Chromatographic analysis was performed using a YMC-Triart C18 chromatographic column, with a gradient elution of mobile phase composed of triethylammonium phosphate and acetonitrile mixture, and a photodiode array detector. HPLC-PDA procedure was selective, linear (in the range from 0.2 to 5.0 µg/mL), accurate (recovery from 92.7 to 115.1 %), precise (RSD from 0.3 to 12.1 %), and with proper detection and quantification limits. The measurement uncertainty was evaluated from validation data, with combined standard uncertainty values of 0.025–0.12 µg/mL (2.1–5.6 %), 0.004–0.28 µg/mL (2.4–12.6 %), and 0.016–0.16 µg/mL (3.2–7.9 %) for His, PCA, and UCA, respectively. Statistical analyses were performed using Monte Carlo simulation and the Mann-Whitney test, as our results were not homoscedastic and deviated from normality. The results indicate that the best audience for quantifying biomarkers were participants up to 35 years old, with all phototypes, and, preferably, female.

Simultaneous Determination of Icotinib, Osimertinib, Aumolertinib, and Anlotinib in Human Plasma for Therapeutic Drug Monitoring by UPLC-MS/MS

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) such as icotinib, osimertinib, and aumolertinib have emerged as promising treatment options for EGFR mutated Non-small cell lung cancer (NSCLC) patients. Additionally, anlotinib, an anti-angiogenic agent targeting VEGFR, FGFR, and PDGFR, has been used in combination with EGFR-TKIs in NSCLC cases. A method utilizing ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed and validated for quantifying icotinib, osimertinib, aumolertinib and anlotinib simultaneously in clinical TDM. The chromatographic separation was performed using a Kinetex C18 column (100mm × 2.1mm) and an elution gradient of ammonium acetate in water acidified with 0.1% formic acid and in acetonitrile. The assay was validated over a linear range of 4–2,000ng/mL for icotinib, 2-1,000ng/mL for osimertinib, 1–500ng/mL for aumolertinib, and 0.8–400ng/mL for anlotinib, following the guidelines on bioanalytical methods by FDA. The quantification method exhibited satisfactory performance in terms of selectivity, accuracy (from 91.3% to 107%), precision (intra- and inter-day coeffficients of variation ranged from 0.944% to 7.48%), linearity, recovery (from 86.0% to 91.9%), matrix effect (IS-normalized matrix factors were from 96.7% to 102%), and stability. Overall, the method proved to be sensitive, reliable, and straightforward, enabling successful simultaneous determination of blood concentrations of icotinib, osimertinib, aumolertinib, and anlotinib in patients. The validity of the method has been confirmed across various instruments.

Validated UPLC-MS/MS method for quantification of melatonin receptor agonists and dual orexin receptor antagonists in human plasma and breast milk: Application to quantify suvorexant and lemborexant in clinical samples

Pharmaceutical care is important for mental health during the perinatal period, which is often characterized by insomnia. In recent years, prescriptions of melatonin receptor agonists (MRAs) and dual orexin receptor antagonists (DORAs) for insomnia have increased; however, their use during the perinatal period has scarcely been reported. In the present study, we developed a UPLC-MS/MS method for the quantification of ramelteon, its metabolite M-II, suvorexant, and lemborexant in human plasma and breast milk to accumulate information on the safety and transfer of MRAs and DORAs into breast milk. Samples of MRAs (ramelteon and M-II) in plasma and breast milk were prepared using liquid–liquid extraction (LLE) with ethyl acetate. For DORAs (suvorexant and lemborexant), LLE with ethyl acetate was applied to plasma samples. For breast milk samples, significant ion suppression was observed for LLE with ethyl acetate. Solid-phase extraction (SPE) cartridges capable of removing phospholipids improved the matrix effects. Finally, protein precipitation with methanol and an SPE cartridge, InertSep® Phospholipid Remover, were selected for breast milk sample preparation. An ACQUITY UPLC BEH C18 column was used for analyte separation. MRAs and DORAs were eluted using isocratic and gradient elution, respectively, and analyzed using electrospray ionization in the positive mode with multiple reaction monitoring. The range of calibration curve for MRAs and DORAs was 0.1–25 and 0.5–50 ng/ml, respectively. Both the plasma and breast milk samples exhibited good linearity over this range. The method was validated by evaluating its accuracy and precision, matrix effect, recovery, carry-over, stability, and dilution integrity. The validated method was successfully applied to clinical samples donated by breastfeeding women and the milk/plasma (M/P) ratio and relative infant dose (RID) of lemborexant (one case) and suvorexant (two cases) were estimated. The M/P ratio of lemborexant was <1, and the RID was 1.05 %. The M/P ratio of suvorexant was <0.1, and RID was 0.11–0.20 %. This method will be useful for future studies evaluating the safety of these drugs during breastfeeding.

A factorial-design/assisted HPLC method for simultaneous monitoring of phenytoin, trimethoprim, and sulphamethoxazole in plasma samples to optimize therapy and mitigate toxicity risks

Phenytoin, an anticonvulsant drug, has a narrow therapeutic index and exhibits significant affinity to plasma proteins, mainly albumin. It is mainly metabolized by cytochrome P450 enzymes 2C9, 2C8, and 2C19. The potential for increased phenytoin toxicity may arise when a drug with enzyme-blocking properties are concurrently administered. The co-administration of trimethoprim is reported to decrease phenytoin clearance by 30 %, therefore, monitoring its plasma concentrations is recommended to minimize the risk of its toxicity and optimize its therapeutic efficacy. This study involves development of a novel and simple HPLC method for concurrent measurement of trimethoprim, sulphamethoxazole and phenytoin in plasma samples. The proposed method was optimized using full factorial design to optimize the variable factors affecting chromatographic responses. The separation was performed via isocratic elution on HyperClone C8 column, using a mobile phase consisting of 30: 38.5: 31.5 (v/v/v) of acetonitrile: 3.3 mM heptane-1-sulphonic acid sodium salt: 0.05 M phosphate buffer containing 0.1 % triethylamine at pH 3.7. The mobile phase was delivered at a flow rate of 1 mL/min with UV detection at 210 nm. The method was validated to verify agreement to the criteria set by the International Council for Harmonization (ICH) and Bioanalytical Methods validation. The results showed excellent linearity over the concentration ranges of 0.5–20.0, 1.0–25.0 and 1.0–20.0 µg/mL with detection limits of 0.41, 0.82 and 0.92 µg/mL for trimethoprim, sulphamethoxazole and phenytoin, respectively.The proposed method was extended for the assessment and of the studied drugs in spiked human plasma samples with % recoveries ranging from 92.60 to 105.30 %, indicating bioanalytical applicability. The proposed method can be utilized to study drug-drug interactions for in-patients therapeutic drug monitoring.

Organic suspension droplet solid-phase dispersive liquid–liquid microextraction ultra-high performance liquid chromatography tandem mass spectrometry determination of antibiotic residues in water samples

A method was developed for the simultaneous detection of 38 antibiotic residues, which included 8 sulfonamides, 14 fluoroquinolones, 9 macrolides, and 7 anthelminthics, in drinking water, tap water, and environmental water. The method involved organic suspension droplet solid-phase dispersive liquid–liquid microextraction, followed by ultra-high performance liquid chromatography tandem mass spectrometry for the analysis of the water samples. Hypersil GOLD C18 chromatography column (100 × 2.1 mm, 1.9 μm) be used for separation of target analytes that were eluted by a gradient mobile phase composition of 0.2 % formic acid–water as mobile phase A and acetonitrile as mobile phase B at a flow rate of 0.2 mL min−1. Using 0.2 mL of octanoic acid as the extractant and 0.3 mL of acetonitrile as the dispersant. Under optimal conditions, the standard curve exhibits a good linear relationship within the range of 1–50 μg L−1. The spiked recovery rate of the method ranges from 63 % to 96 %, with a relative standard deviation of 0.6 % to 7.3 % (n = 6). The detection limit of the method is between 0.1 and 0.2 μg L−1. This method is characterized by its simplicity, speed, and high sensitivity, which sets the groundwork for investigating the exposure levels and environmental behavior of antibiotics.

Advanced stability-indicating gradient elution reverse phase-high performance liquid chromatography method development and validation for simultaneous estimation of rutin and curcumin using an analytical quality by design approach

This research delves into a systematic Analytical Quality by Design (AQbD) approach to develop and validate a stability-indicating robust RP-HPLC method for the simultaneous determination of Rutin and Curcumin, compounds known for their anti-inflammatory, antioxidant, and wound-healing properties. Utilizing QbD principles, critical method parameters (CMPs) were identified and screened using design of experiments (DoE) and optimized via response surface methodology (RSM) with Central Composite Design (CCD). The optimized chromatographic separation used a Phenomenex Luna C18 column with gradient elution, using methanol and 0.1% formic acid, a flow rate of 1 ml/min, and detection at 345 nm (isosbestic point). The retention times were 5.63 mins for Rutin and 17.01 mins for Curcumin. The stability indication of the developed HPLC method was performed, and validation parameters were meticulously conducted following the ICH Q2(R1) guidelines, demonstrating linearity over 2-10 μg/ml for Rutin and 5-25 μg/ml for Curcumin, with correlation coefficients of 0.9999 and 0.9998, respectively. Precision studies showed %RSD within acceptable limits, and accuracies ranged between 97-100%. This validated HPLC method offers a reliable, sensitive approach for simultaneous quantifying Rutin and Curcumin, suitable for routine analysis in pharmaceutical laboratories and quality control.

Preparation of two amphiphilic dendritic small molecule gelators based on poly (aryl ether) modified silica-based chromatographic stationary phases and molecular shape recognition for shape-restricted isomers

Geometric isomers tend to have similar polarities and differ only in molecular shape. Vigorously developing new stationary phases to meet the requirements for the separation of isomers that have similar physicochemical properties is still an urgent topic in separation science. Poly (arylene ether)-based dendrimers are known for their multifunctional branched peripheral structures and high self-assembly properties. In this paper, two amphiphilic dendritic organic small molecule gelling agents based on poly (aryl ether), PAE-ANT and PAE-PA, were prepared and conjugated to the silica surface. SiO2@PAE-ANT and SiO2@PAE-PA were used as HPLC stationary phases for the separation of non-polar shape-restricted isomers. Both stationary phases have very high molecular shape selectivity for isomers such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), tocopherols and carotenoids. Separation of cis-trans geometric isomers such as diethylstilbestrol and polar compounds such as monosubstituted benzenes and anilines can also be achieved. These two columns offer more flexible selectivity and higher separation performance than commercial C18 and phenyl columns. There is a difference in molecular shape selectivity between the two stationary phases for the same analyte test probes. SiO2@PAE-ANT showed slightly better linear selectivity for non-polar shape-restricted isomers compared to SiO2@PAE-PA with Janus-type PAE-PA bonding phase. This separation behavior may be attributed to the ordered spatial structure formed by the gel factor on the surface of the stationary phase and the combined effect of multiple weak interaction centers (hydrophobic, hydrophilic, hydrogen bonding and π-π interactions). It was also possible to separate nucleoside and nucleobase strongly polar compounds well in the HILIC mode, suggesting that hydrophilic groups in PAE-ANT and PAE-PA are involved in the interactions, reflecting their amphiphilic nature.The results show that the ordered gelation of dendritic organic small molecule gelators on the SiO2 surface, along with multiple carbonyl-π, π-π and other interactions, play a crucial role in the separating shape-restricted isomers. The integrated and ordered functional groups serve as the primary driving force behind the exceptionally high molecular shape selectivity of SiO2@PAE-ANT and SiO2@PAE-PA phases. Alterations in the structure of dendritic organic small molecule gelators can impact both molecular orientation and recognition ability, while changes in the type of functional groups influences the separation mechanism of shape-restricted isomers.

A robust ultra performance liquid chromatography method for dacarbazine quantification in nanosponge formulation: A quality by design approach

A new, user-friendly Ultra Performance Liquid Chromatography (UPLC) method was developed using Analytical Quality by Design (QbD) for precise analysis of Dacarbazine in both bulk powder and Dacarbazine-loaded Nanosponges. This method follows ICH guidelines and uses a statistically designed approach to optimize the separation conditions for Dacarbazine. A central composite design was implemented to identify the ideal combination of organic solvent (mobile phase composition) and flow rate. The UPLC method was successfully optimized for rapid separation (1.3 minutes) for Dacarbazine using a mobile phase consisting of 70% Methanol: 30% Orthophosphoric acid (0.1%) at a flow rate of 1.0 mL/min, using a Hypersil C18 column. The method exhibited excellent linearity with a correlation coefficient value of 0.997 across a concentration range of 4-20 μg/mL, accuracy (drug recovery of 96-100%) and sensitivity (LOD 1.15 μg/mL, LOQ 3.50 μg/mL) and it successfully quantified the drug in the formulated Dacarbazine-loaded Nanosponges. This QbD-driven UPLC method offers a valuable tool for reliable Dacarbazine quality control in pharmaceuticals providing rapid analysis, high accuracy, and low detection limits.

Quantitative analysis of mixed lipid nanostructures in rat skin by HPLC-MS

Abstract Liquid chromatography-mass spectrometry (LC-MS) is a very sensitive technique for determining small concentrations of drugs in fixed dose combinations or even those deposited in skin layers. Therefore, an LC-MS method was applied for determining the drugs under investigation, namely, clioquinol (CLIO), tolnaftate (TOL), and betamethasone (BETA) in Quadriderm® cream and mixed lipid nanostructures (MLNs) prepared in laboratory in the presence of potential interferents, and was applied as a dermato-kinetic study in rat’s skin. The separation was achieved within 4.5 min by using C18 column as a stationary phase and the mobile phase used were 20% phase A composed of 0.1% formic acid (v/v) and 80% phase B composed of 0.1% formic acid in acetonitrile (v/v), coupled with triple quadrupole mass spectrometer. MLNs were prepared and characterized to be compared with the conventional commercially available Quadriderm® cream. The proposed method was accurate and precise with a linearity range of 0.2–20.0 µg·mL−1 for BETA, and 0.5–400.0 µg·mL−1 for CLIO and TOL and a better bioavailability of the new formulation was obtained ensuring the capability of the nanoparticles to accumulate the drugs within the skin layers. In conclusion, the LC-MS method was accurate and precise for the determination of the three drugs under investigation in cream dosage form and skin tissues.

Determination of Gb3 and Lyso-Gb3 in Fabry Disease-Affected Patients by LC-MRM/MS

Limited or absent activity of the enzyme α-galactosidase A (α-Gal A), due to mutation in the related gene on the X chromosome, leads to the development of a rare hereditary and genetic disease known as Fabry disease (FD). This pathology involves a progressive accumulation in various organs of the substrates of the enzyme e.g., globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (Lyso-Gb3), suggesting these molecules as biomarkers of Fabry disease. The present paper describes the development of an analytical strategy for the identification and quantification of Gb3 and Lyso-Gb3, in serum and blood samples by using liquid chromatography (LC) coupled to mass spectrometry in multiple reaction monitoring (MRM/MS) ion mode. The best experimental conditions were obtained by extracting the glycolipids with chloroform/methanol/H2O (2/1/0.3) and by separating them on a C4 column with a linear gradient (A: H2O with 2 mM ammonium formate. B: methanol with 1 mM ammonium formate, both acidified with 0.2% formic acid). The best transitions (a combination of precursor and fragment ions—m/z) were 786.8 m/z &gt; 268.3 m/z for Lyso-GB3, 1137.3 m/z &gt; 264.3 m/z for Gb3, 1039.3 m/z &gt; 264.4 m/z for N-heptadecanoyl-ceramide trihexoside, and 843.5 m/z &gt; 264.3 m/z for N-glycinated lyso-ceramide trihexoside, the latter being used as an internal standard. The developed method provided a reliable, fast, and effective procedure for direct measurements of GB3 and Lyso-GB3 in serum and blood for diagnosis of Fabry disease, suggesting this method as a complementary assay to the current enzymatic test. Therefore, this approach could open new insights into the clinical diagnostics of lysosomal storage disorders.