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Aim: An accurate and fast ultra-high performance liquid chromatography coupled with tandem mass spectrometry analytical method was developed and validated for quantifying fluconazole levels in human plasma according to the US FDA guidelines.Materials & methods: A simple protein precipitation by acetonitrile was employed for the sample preparation. The chromatographic separation was carried out using isocratic elution of water (0.1% formic acid) and acetonitrile on an Acquity ultra-high performance liquid chromatography HSS T3 column. Samples from ten adult patients diagnosed with candidemia who received fluconazole treatment were analyzed.Results & conclusion: The method demonstrated excellent linearity and stability within the 1-50 µg/ml range (r2 >0.999). The intraday and interday precision were determined with coefficient of variation values ranging from 1.4 to 4.38% and 2.8 to 6.6%, respectively. This rapid and selective method has successfully analyzed 27 plasma samples. The straightforward sample preparation in a single step and the reduced analysis time make this method suitable for adult patients with candidemia, leading to improved clinical outcomes.

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BACKGROUND: Medicinal plants have curative properties due to the presence of various complex chemical substances of different compositions, which are found as secondary plant metabolites in one or more parts of the plants. Moringa oleifera from Moringaceae and Beta vulgaris root are, native to India, grows in the tropical and subtropical regions of the world. It is commonly known as 'drumstick tree' or 'horseradish tree' or 'miracle tree'. Incorporation of more herbal powder leads to much complexity. Above plants were chosen for their utmost nutritional values. RESULTS: Herbal tablet and granules were prepared and evaluated further for various Physico-chemical parameters as a nutritional supplement. Promising results indicate that prepared formulations have potential as supplements. CONCLUSIONS: Present communication mainly focused on estimation of marker components by Reverse Phase-High Performance Liquid Chromatography. It showed the presence of enough number of secondary metabolites and minerals which can be easily consumed by all age groups.

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BACKGROUND AIMS: The administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. METHODS: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. RESULTS: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314-570 wells) based on the laboratory-specific CFE (42-76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. CONCLUSIONS: The D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.

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In this study, an alternative method to compendial analytical procedures with enhanced detection and separation capabilities was validated for the quality assessment of glutathione (GSH) drug substance. The related impurities A, B, C, and D present in GSH drug substance were characterized using a one-dimension proton nuclear magnetic resonance (1D 1H NMR) method on a 600 MHz spectrometer equipped with a liquid nitrogen cryoprobe. Two sample preparations at different pH were optimized to ensure the unambiguous identification of different impurities in the GSH samples. Specifically, impurities A and C in a GSH sample can be tested at pH 3.0, while pH 7.4 is more suitable for testing impurities B and D. The quantitative NMR (qNMR) method was validated following International Council for Harmonisation (ICH) guidelines. The limit of detection (LOD) was less than 0.1% wt for an individual impurity, and the limit of quantitation (LOQ) ranged from 0.14 to 0.24% wt, using about 14 min experimental time per spectrum. Following validation, the qNMR method was applied to assess different commercial GSH bulk substance samples, an in-house compounded GSH drug product, and a GSH dietary supplement product. The method was also applied to monitor GSH degradation (hydrolysis and oxidation) over time to provide quantitative information on GSH degradation and stability. The results suggest that the qNMR method can serve as a highly specific and efficient orthogonal tool for assessing the quality of GSH pharmaceuticals, providing both qualitative and quantitative information on GSH and its related impurities A-D.

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The chemical composition of Nonea pulmonarioides extracts were investigated for the first time. The phytoconstituents of the methanol extracts were screened by using LC/MS-MS technique. The anticancer activity of the acetone and methanol extracts were measured against four cancer cell lines; MCF-7, PC3, HT-29, and U-87 MG. Thirty phenolic compounds were identified, rosmarinic (90.06 mg analyte/g extract) and fumaric acids (39.737 mg analyte/g extract) were major compounds of the studied species. Moreover, both methanol and acetone extracts were found to have strong anticancer activities. The acetone extract HT-29 (with IC50 of 10.17 ± 0.25 µg/mL) compared with standard cis-platin (with IC50 of 22.20 ± 0.72 µg/mL) with apoptotic mediated programmed cell death. These findings identified N. pulmonarioides as a potential species exhibiting anticancer properties. In conclusion, the compelling results show that the methanol extract contains possible bioactive compounds with anticancer properties that require isolation and further characterisation.

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Oligosaccharides are significant in mammalian milk, where they serve as prebiotics that promote the growth of beneficial gut bacteria in infants. Comprehensive research of milk oligosaccharides requires precise and validated analytical methods for compositional studies. To address this need, the focus of our study was to develop and validate an analytical method using UPLC-MS/MS to quantify seven specific oligosaccharides found in mammalian milk. The developed and optimized method has adequate linearity, accuracy, and precision parameters. The detection (LOD) and quantification (LOQ) limits for the seven compounds ranged from 0.0018 to 0.0030 µg/mL and 0.0054-0.0063 µg/mL, respectively. The sample preparation method yielded recovery rates above 90.5 %. Furthermore, no significant matrix effect was observed. The validated method was successfully applied to human, goat, and bovine milk samples, demonstrating its proficiency in identifying variances in the concentration of oligosaccharides across different mammals. This versatile method will allow future research about factors affecting oligosaccharide composition.

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The research study aimed at providing an accurate low-dose benzene exposure assessment method, by validating diffusive monitoring techniques for benzene personal exposure measurements at workplaces where benzene concentrations are expected in the low ppb range, such as in the present-day chemical, petrochemical, foundry, and pharmaceutical industry. The project was aimed at addressing the need for a robust and fully validated method to perform personal exposure measurements considering that the occupational exposure limit value for benzene is going to be significantly lowered in the next few years. Diffusive sampling offers a reliable alternative to pumped sampling methods, intrinsic safety in potentially explosive atmospheres, lightness, and ease of use. In this study, the radiello® diffusive sampler, with the packed activated charcoal RAD130 adsorbing substrate [suitable for solvent desorption and analysis by high-resolution gas chromatography-flame ionization detection (HRGC-FID)], was used. The experiments have been conducted following the ISO 23320 standard in the range from 0.005 to 0.1 ppm (16 to 320 µg/m3), yielding a full validation of the sampling and analytical method. The sampler performances have fulfilled all requisites of the ISO 23320 standard, in particular: bias due to the selection of a non-ideal sorbent is lower than 10% (no significant back diffusion of benzene due to concentration change in the atmosphere); bias due to storage of samples for up to 2 months is lower than 10%; nominal uptake rate for benzene on RAD130 is 74.65 mL/min; and expanded uncertainty of the sampling and analytical method is 20.6%. The sampling and analytical method is therefore fit-for-purpose for the personal exposure measurements aimed at testing compliance with occupational exposure limit values for benzene. The method is also fit for short-duration exposure monitoring related to specific tasks, and other volatile organic compounds, usually found in the same workplaces, such as aliphatic and aromatic hydrocarbons and some oxygenated compounds, have also been studied. In particular, n-hexane and isopropyl benzene, whose classification is currently under revision, can be efficiently monitored by this technique.

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The diphenolic diterpene carnosol was isolated from several species of the family Lamiaceae, including Lepechinia mutica, a medicinal plant endemic to Ecuador. The compound has exhibited high antioxidant, anti-inflammatory, antimicrobial, neuroprotective, and antifungal properties, as well as promising cytotoxicity against prostate, breast, skin, leukemia, and human colon cancer cell lines. In this paper, we developed and validated a simple, accurate, and reliable analytical HPLC-UV-ESI-IT-MS method, carried out on a C18 column, which is potentially suitable to quantify carnosol in plant extracts. The procedure complied with the established ICH validation parameters of analytical range (linearity in the range of 0.19-5.64 µg/g dried leaves; REAVERGE = 4.9%; R2 = 0.99907), analysis repeatability (RSD = 2.8-3.6%), intermediate precision (RSD = 1.9-3.6%), accuracy (estimated as % carnosol recovery in the range of 81 to 108%), and robustness. Finally, the LOD (0.04 µg/mg) and LOQ (0.19 µg/mg) values of carnosol/dried leaves were determined. Using this validated method, the content of carnosol in L. mutica was estimated to be 0.81 ± 0.04 mg/g of dried leaves (0.081%).

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Many people in developing countries rely on herbal remedies for their primary healthcare needs. The challenge however is that several of these products lack proper documentation of quality and safety. To ensure consistent quality, validated methods are needed to establish and control quality attributes associated with identity, purity, and levels of bioactive constituents of the respective herbal materials. The present study focused on Phyllanthus urinaria (PU), a widely used medicinal plant in Ghana and West Africa that lacks the necessary quality control standards. The study aimed to develop an HPTLC identification method, which together with UPLC-ESI-Q-TOF-MS/MS analysis established the identity of PU samples and differentiated PU from other closely related Phyllanthus species. Quantitative UPLC and HPTLC methods were developed to assess the contents of selected active markers in the PU samples, which invariably led to the proposal of acceptance criteria for the active markers. Prior to the content analyses, the sample extraction procedure was optimized through the use of Design of Experiment method. The effects of harvest time and geographic origin on the content of active compounds were demonstrated in the investigations. PU samples were also found to be contaminated with higher levels of pesticides like chlorpyrifos and folpet. Essentially, this study provides analytical protocols, insights into the quality status of PU samples in Ghana, and analytical specifications contained in a drafted monograph for future consideration in regional and subregional African pharmacopoeias.

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Aim: To investigate the stability of the anti-pneumococcal (PCP) and anti-haemophilus type B (Hib) immunoglobulins (IgGs) in human IgG-depleted serum samples frozen at -20°C. Materials & methods: Modified commercially available immunoassays (ELISAs) were bioanalytically validated. These ELISAs were used to measure levels of the two anti-bacterial IgG in samples kept at -20°C for up to 15 months. Human IgG-depleted serum was spiked with GAMMAGARD Liquid to obtain those samples. Results: Both ELISAs passed the validation test. Anti-PCP IgG and anti-Hib IgG were shown to be stable for at least 15 months at -20°C. Conclusion: These data confirm the stability of anti-bacterial IgG in human IgG-depleted serum and support the common practice of testing frozen samples.

Immunodeficiency disorders can prevent your body from fighting infections. These disorders make it easier to catch viruses and bacterial infections caused by so-called pathogens. Patients suffering from immunodeficiencies are treated throughout their lives with antibodies purified from human plasma. This immunoglobulin replacement therapy, which helps to avoid infections, provides specific antibodies directed against these pathogens. An antibody is a protein produced by the body's immune system to detect (bind) antigens and to help eliminating harmful substances. Little is known about the stability of such specific antibodies in samples taken from patients during clinical studies carried out to improve the replacement therapy. We investigated the stability of two such antibodies using a standard technique for their measurement. In a process termed validation, these methods were demonstrated to deliver accurate and precise results. For the stability study, we prepared human serum (= the liquid part of human blood) samples with specific antibodies levels expected in samples from patients on replacement therapy. These samples were kept frozen at -20°C for up to 15 months. The data obtained on analysis of the frozen samples showed the adequate stability of both antibodies directed against important pathogen. This stability confirms a common testing practice applied for samples obtained in clinical studies where usually such samples are not tested immediately but are stored frozen and tested in batches. In particular, the data for the two anti-bacterial antibodies support the storage of such samples for at least 15 months at -20°C before testing.

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The release of hazardous chemicals into aquatic environments has long been a known problem, but its full impact has only recently been realized. This study presents a validated liquid chromatography-mass spectrometry (HPLC-MS/MS) method for detecting pharmaceutical and pesticide residues in mussels (Mytilus galloprovincialis). An innovative MS-compatible extraction method was developed and validated, demonstrating successful recovery rates for analytes at three different concentration levels (25-95%). The method detected the target analytes at ng/g concentrations with high accuracy (-7% to 11%) and low relative standard deviation (<10%) for both intra-day and inter-day analyses. After validation, the method was applied to mussel samples collected from a commercial farm near Senigallia, Adriatic Sea, detecting different contaminants in the range of 2-40 ng/g (dry weight). The study provides a valuable tool for investigating the potential threats posed by diverse contaminant classes with high annual tonnage, including analytes with known persistence and/or illegal status.

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A bioanalytical LC-MS/MS method was developed and validated to determine ceftaroline in microdialysate samples from plasma and brain. Ceftaroline was separated using a C18 column and a mobile phase consisting of water and acetonitrile, both with 5 mM of ammonium formate and acid formic 0.1%, eluted as gradient. Ceftaroline was monitored using electrospray ionization operating on positive mode (ESI+) monitoring the transition 604.89 > 209.3 m/z. The method showed linearity in the concentration range of 0.5-500 ng/mL for brain microdialysate and 0.5-2500 ng/mL for plasma microdialysate with coefficients of determination ≥0.997. The inter-and intra-day precision, the accuracy, and the stability of the drug in different conditions were in accordance with the acceptable limits determined by international guidelines. Plasma pharmacokinetics and brain distribution of the drug were carried out after intravenous administration of 20 mg/kg of ceftaroline to male Wistar rats. The estimated geometric mean (geometric coefficient of variation) area under the curve (AUC0-∞) was 4.68 (45.8%) mg·h/L and 1.20 (54.2%) mg·h/L for plasma and brain, respectively, resulting in a brain exposure of about 33% (AUCfree brain/AUCfree plasma). The results indicate that ceftaroline presents good penetration in the brain when considering free plasma and free brain concentrations.

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A capillary zone electrophoretic (CZE) method was developed, validated, and applied for the assay of metformin (MET) and pioglitazone (PIO) in pharmaceutical formulations. The optimum running buffer composition was found to be 75 mmol/L phosphate buffer containing 30% acetonitrile (ACN) at pH 4.0. The optimum instrumental conditions were found to be injection time, 10 s; applied voltage, 25 kV; hydrodynamic injection pressure, 0.5 psi for 10 s, capillary temperature, 25 °C; and the detection wavelength, 210 nm. The quantifications were calculated based on the ratio of the peak areas of analytes to atenolol as an internal standard. The CZE method was validated in terms of accuracy (98.21-104.81%), intra- and inter-day precision of migration time and peak area (relative standard deviation ≤ 5%), linearity (correlation coefficients ≥ 0.9985), limit of detection (≤0.277 µg/mL), and limit of quantitation (≤0.315 µg/mL). The proposed method was applied for the analysis of PIO and MET both individually and in a combined dosage tablet formulation. All electrophoretic parameters were calculated and evaluated. A previously reported high-performance liquid chromatographic (HPLC) method was also applied to the same samples. A comprehensive comparison was then carried out for the analytical features of both methods CZE and HPLC. Comparable results were obtained with the advantage of reagent consumption and separation efficiency of CZE over HPLC and shorter analysis time by HPLC compared with CZE.

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Pregnenolone (PREG) is an endogenous steroid frequently sold as an over-the-counter dietary supplement touted to promote neurological and immunological health. While the PREG dietary supplement is added to the diet for health benefits, there are no FDA approved PREG drugs. However, compounded PREG drug products are available to U.S. patients. The FDA works with state regulatory authorities on the oversight of compounding activities, including developing 503A and 503B lists of bulk substances that compounders are permitted to use. PREG is one of the substances publicly nominated to be included on the 503B list. Compounded hormone therapies such as those using PREG are of interest given the lack of standardization in compounded drug products which may increase the possibility of underdosing, overdosing, or contamination. However, no USP monograph currently exists to evaluate the quality of PREG drug substance or product. To address knowledge gaps and assist in quality control, a simple and rapid quantitative proton nuclear magnetic resonance spectroscopy (qNMR) method for the identification and assay of PREG in different types of PREG products was developed and validated. PREG samples were characterized using 1D 1H and 2D 1H-13C HSQC NMR spectra. The qNMR assay method (taking approximately 10 min per NMR spectrum) was validated for precision, accuracy, specificity, robustness and linearity per ICH Q2(R1) guidance. The method was validated in a range from 0.032 to 3.2 mg/mL. As a proof of concept, seven PREG bulk substance samples, three tablet and two capsule PREG dietary supplements were assessed by the qNMR analytical procedure. NMR data from all tested samples met the expected criteria for identification and assay. The results demonstrate the potential of qNMR for the quality assessment of different types of PREG samples.

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Cherries are popular fruits due to their health benefits, organoleptic quality, and attractive appearance. Since highly polar pesticides are of low mass and amphoteric character, and are not amenable to traditional multi-residue extraction methods, they are more commonly not included in the pesticide monitoring program. This study aims to determine twelve highly polar pesticide residues in cherry samples intended for export from Turkey. A total of 16,022 cherry samples from 2018−2020 harvests in four production areas of Turkey were analyzed using a modification of the Quick Polar Pesticides method and liquid chromatography-tandem mass spectrometry. The method was validated at two fortification levels (0.01 and 0.05 mg kg−1), and good recoveries (87.4−111.4%) and relative standard deviations (<6%) were achieved for all analytes. The limits of quantification were in the range of 1.08−2.55 µg kg−1. Overall, 28.4% of the analyzed cherry samples were detected with phosphonic acid, calculated as fosetyl aluminium (fosetyl-Al) in amounts up to 77.7 mg kg−1. For 2304 samples (14.4%), the residues exceeded the European Union maximum residue level of 2 mg kg−1. There is no reason to be concerned about long-term exposure to phosphonic acid/fosetyl-Al, and the other highly polar pesticides through the consumption of sweet cherry.

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A targeted UHPLC-MS/MS isotopic dilution method has been developed for the simultaneous quantification of 18 different free and protein-bound aromatic amino acid oxidation products in food and biological matrices. All analytes, including critical isomeric pairs of Tyr, o-Tyr, m-Tyr, and dioxyindolylalanine diastereomers were chromatographically resolved to obtain high selectivity, without the need for derivatizing or ion pairing agents. The results of method validation showed adequate retention time reproducibility [0.1-0.6% coefficient of variation (CV) for over 224 injections], accuracy (within ±1-20% of the nominal concentration), and precision (1-17% CV) for all target analytes. The lower limit of quantification was calculated in different matrices using both the Hubaux-Vos approach and accuracy and precision data showing values in the range of 0.2-15 ng/mL. Use of stable isotope-labelled internal standards compensated errors due to matrix effects and artefactual degradation of analytes. Both acid and enzymatic hydrolyses were tested to obtain the best possible results for the quantification of protein oxidation products, demonstrating the stability of target analytes under hydrolytic conditions. The method was successfully applied to quantify target analytes in serum, tissue, milk, infant formula, pork liver pâté, chicken meat and fish. The method was also applied to assess the role of Fenton's reagent in oxidizing Trp, Phe and Tyr residues in different proteins, with results showing o-Tyr, dioxyindolylalanine diastereomers, kynurenine, dityrosine being the main oxidation products. The Fenton chemistry favored the formation of o-Tyr over m-Tyr from Phe with 2-36 folds higher yields. 3-Nitrotyrosine, a marker of protein nitration, was also detected in samples treated with Fenton's reagent.

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Bio-analytical, nano-quantitative spectrofluorimetric estimation of two non-classical ß-lactam antibiotics; meropenem (MP) and ertapenem (EP) is presented. The method is based on the enhancement of the fluorescence intensity of MP-Eu3+/EP-Eu3+ with silver nanoparticles (AgNPs). AgNPs were synthesized and characterized by UV and transmission electron microscope (TEM). The plasmon resonance produced an intense absorption maximum at 398.0 nm. TEM micrograph showed the particle morphology with an average particles size of 13.0 ± 2.95 nm. The fluorescence intensities were measured against blank reagents at λem of 396.0 nm and 405.0 nm after excitation at λex 305.0 nm and 303.0 nm for MP and EP, respectively. Under optimum conditions, the relative fluorescence intensity showed a good linear relationship with the concentration ranges of 4.0-14.0 and 4.0 -12.0 ng/mL with excellent correlation coefficients of 0.9998 and 0.9997, and limit of detection of 0.84 and 0.86 ng/mL for MP and EP, respectively. The method was successfully applied for direct analysis of MP and EP in their drug substances and pharmaceutical vials. The significant, sensitivity and practicality of the method facilitated MP detection in real plasma samples. Bio-analytical validation was performed according to FDA. The method was rectilinear over the ranges of, 5.0 -75.0 µg/mL plasma. Interestingly, this described system has a promising benefit for various applications exploiting the dramatically enhanced-fluorescence occurrence.

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This research aimed at developing an analysis method, which was optimized and validated to determine the content of mercury in skin lightening cream discovered in the market in Bandar Lampung, Indonesia, through the use of microwave plasma atomic emission spectroscopy (MP-AES). The optimization on the analysis method was conducted on pump rate, viewing position, and reductant concentration in order to obtain the highest mercury emission intensity, while the solution stability was optimized to know the stability of mercury in the solution. The result showed that the method developed had precision with a relative standard deviation of 2.67%, recovery value of 92.78%, and linearity with an r value of 0.993, respectively. The sensitivity of the instrument detection had a limit of analysis method detection and quantification of 0.59 and 1.98 µg/L, respectively. The results of the test of the lightening cream (8 of 16 samples) positively contained mercury in the range of 422.61-44,960.79 ng/g. Therefore the method of analysis developed may be used for routine analysis of chemicals in any cosmetics products.

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Tazarotene, a member of the acetylenic class of retinoids, is a third-generation prescription topical retinoid sold as a cream, gel, or foam. In its gel or cream formulations, tazarotene is at a concentration of either 0.1 % or 0.05 %. Fabric phase sorptive extraction membranes are used to selectively isolate and preconcentrate target analytes from different sample matrices. In this study, the tazarotene gel formulation was directly applied to fabric phase sorptive extraction membranes and extracted through a mixture of acetonitrile and water (80:20, v/v) to obtain a clean product free of colloidal suspension of tazarotene gel formulation. The final solutions were injected into an HPLC system equipped with a Zorbax 5 µm Phenyl-Hexyl LC Column (250 × 4.6 mm). Injection volume was 50 µL and UV detection was performed at 326 nm. The flow rate was 1.0 mL min-1 while using an isocratic elution with a mixture of ammonium acetate (50 mM) and methanol (15:85, v/v) as the mobile phase. The method was validated according to ICH guideline Q2 (R1) and successfully applied to gel formulations including 0.01 % tazarotene. This is the first reported application of fabric phase sorptive extraction in the analyses of gel formulations. The capability of fabric phase sorptive extraction membranes to clean up the sample matrix and prepare active pharmaceutical ingredients to be analyzed with acceptable recovery (>98.0 %) and reproducibility may encourage quality control laboratories to use fabric phase sorptive extraction in routine applications.

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