Complete Sample Analysis Research Articles

Caffeine in Tablet Dosage Form: Analytical Method Development and Validation by Using U.V. Spectroscopy

Abstract: Caffeine (1,3,7-trimethylpurine 2,6 dione) is a CNS stimulant. Caffeine is used as a drug having drug-addicted properties having a U.V. cutoff in the range of 272 nm to 297 nm in the U.V. spectrum. In this paper, we took a tablet with Caffeine as a single dose in solid dosage form with excipients that do not have a sharp peak in the U.V. spectrum range from 200 nm to 400 nm. The solvent used as a vehicle is a simple polar solvent with a U.V. cutoff at around 189nm to 191 nm. The U.V. spectrophotometer that we used is of Shimdzu having a double beam, which has two provisions to keep reference and sample at a time to complete sample analysis. Firstly we make a calibration curve of Caffeine with a polar solvent. For the research, development, and production of medicines, analytical technique related to creation and validation are essential. Therefore, in this study, a readily reproducible, straightforward, and quick technique of U.V. spectrophotometric quantitative measurement of Caffeine in the form of tablets is adopted. According to ICH criteria, the parameters to be examined include linearity, specificity, accuracy, precision, and robustness. Purified/deionized water was used as the diluent for the drug quantification at the wavelength of 248.5 nm. Beer-law Lambert's applies to Caffeine at concentrations between 10 and 50 g/ml. Recovery was used to test the method's accuracy, and the results ranged from 99.46 to 100.67%. This approach was used for routine analysis of Caffeine in tablet dosage form.

Abstract 2948: A multimodal, label-free tool for gentle and efficient cell separation

Abstract LevitasBio has developed the LeviCell™ platform, a powerful new technology for cell separation and characterization that utilizes magnetic fields to levitate cells. Unlike other methods, cell separation via magnetic levitation does not require dyes, antibodies or specific markers, and the cells are not modified or perturbed in any fashion. However, magnetic levitation also does not preclude the use of these tools to further enhance separation of cells. In its simplest form, magnetic levitation requires only the addition of an inert paramagnetic compound to the media in which the cells are suspended. The cells are subsequently introduced into an externally applied magnetic field while contained within a single-use cartridge, which causes the cells to levitate in solution. The cells’ intrinsic properties, including density and magnetic susceptibility, determine the height to which the cells levitate. As different cell types often have different such properties, they may often levitate to different heights, facilitating their separation. Continuous imaging allows for complete sample analysis and characterization with the benefit of real-time control over the cell separation. The simplicity of magnetic levitation enables cells to be treated gently without use of high pressure or other perturbations that commonly lead to increased cellular stress responses, specific cell type activation, or even cell death. The direct and gentle flow path leads to high yields of live cells even with very low input cell numbers. This novel cell separation technology enables a variety of applications, including: the enrichment of viable cells from dead/dying cells and debris; separation of different cell types possessing distinct densities; and, in cases where the intrinsic density changes, the separation of differentiated from undifferentiated cells. Given that the levitation chamber is flanked by two magnets on the top and bottom, the levitation technology also inherently allows for the use of magnetic particles to deplete undesired cells within a cell population. Here we illustrate the performance of anti-CD45 antibody-conjugated magnetic particles for CD45+ cell depletion with the LeviCell system. CD45+ depletion rates of greater than 99% are readily obtained across a wide range of total CD45+ input cell numbers, from 105 to 107 total cells. Furthermore, CD45+ cell depletion is performed simultaneously with live cell enrichment, allowing for the collection of a high viability CD45- live cell population. Citation Format: Kevin Travers, Chris Stevens, Susana Jett, Suzanne Weaver, Teddy de Groot, Stefan Ha, Geoff Facer, Kathy Lazaruk. A multimodal, label-free tool for gentle and efficient cell separation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2948.

Simultaneous and Direct Estimation of Glymes in Pharmaceuticals and Biopharmaceuticals by Headspace Gas Chromatograph Coupled with Mass Spectrometer

The analytical approach used for the effective trace-level determination of monoglyme and diglyme in pharmaceuticals and biopharmaceuticals employs a simple sample preparation technique. This method involves headspace gas chromatograph coupled with mass spectrometer and is based on an oven scheduling approach which uses helium gas as the mobile phase; a complete sample analysis is conducted in less than 1h which does not include the injection time of standard replicates. The detection and quantitation limits of this advanced technique are 0.01 and 0.03μg/mL for both monoglyme and diglyme, respectively. The successful separation of monoglyme and diglyme was confirmed by determining their corresponding specific molecular masses and its fragments. This method is precise, sensitive, accurate, linear, and robust as determined by method validation performed according to the International Council on Harmonization validation guidelines Q2 (R1) and United States Pharmacopoeia. In this study, monoglyme and diglyme were not detected in the samples of pharmaceuticals and biopharmaceuticals when using this technique. By headspace chromatograph coupled with mass spectrometer, monoglyme and diglyme can be easily estimated at trace levels during the commercial preparation of biologically and chemically active substances in both the biopharmaceutical and pharmaceutical industries. The main benefit of this research work is to get the GC-FID and GCMS data simultaneously to determine monoglyme and diglyme at micro-level in Pharmaceuticals and Biopharmaceuticals.

NATURAL RESOURCES DEPLETION, RENEWABLE ENERGY CONSUMPTION AND ENVIRONMENTAL DEGRADATION: A COMPARATIVE ANALYSIS OF DEVELOPED AND DEVELOPING WORLD

This article investigates the impact of renewable energy consumption and natural resource depletion on environmental degradation from 1990 to 2014. The analysis of this study is distributed into three parts, developing country analysis, developed country analysis and complete sample analysis. An insignificant relation has found between natural resource depletion and environmental degradation in the case of complete sample analysis and developing country analysis, but vica-versa in developed countries. Fossil fuel energy consumption has a positive and significant impact on environmental degradation in developing countries. Renewable energy consumption has negative impact on environmental degradation in the case of complete sample analysis and developed country analysis, but visa-versa in developing countries. Economic growth positively and significantly effecting environmental degradation in all the three cases, this mean for higher economic growth we have to bear some environmental degradation. But it is the need of the hour that we should find some threshold between economic growth and pollutant emissions, so that a healthy environment can be safe for coming generations. So, for a healthy environment, fossil fuel consumption should be reduced and consumption of renewable energy with merchandised trade and urbanization can be encouraged.

Gateway or common liability? A systematic review and meta-analysis of studies of adolescent e-cigarette use and future smoking initiation.

Studies have consistently found a longitudinal association between e-cigarette use (vaping) and cigarette smoking. Many have interpreted such association as causal. This systematic review and meta-analysis evaluated the plausibility of a causal interpretation by (1) estimating the effect of adolescent vaping on smoking initiation, adjusted for study quality characteristics, (2) evaluating the sufficiency of adjustment for confounding based on the social development model (SDM) and the social ecological model (SEM) and E-value analyses and (3) investigating sample attrition and publication bias. Systematic review and meta-analysis of longitudinal studies that examined the association between e-cigarette use at baseline and smoking at follow-up. Participants were non-smokers aged <18 at baseline. Meta-analysis of 11 studies showed a significant longitudinal association between vaping and smoking [adjusted odds ratio (aOR)=2.93, 95% confidence interval (CI)=2.22, 3.87]. Studies with sample sizes <1000 had a significantly higher odds ratio (OR=6.68, 95% CI=3.63, 12.31) than studies with sample sizes>1000 (OR=2.49, 95% CI=1.97, 3.15). Overall, the attrition rate was very high (median=30%). All but one study reported results from complete sample analysis, despite those dropping out having higher risk profiles. Only two studies comprehensively adjusted for confounding. The median E-value was 2.90, indicating that the estimates were not robust against unmeasured confounding. There is a longitudinal association between adolescent vaping and smoking initiation; however, the evidence is limited by publication bias, high sample attrition and inadequate adjustment for potential confounders.

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW < 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 ºC. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW < 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 ºC. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

Abstract P190: Validity and Reproducibility of a Food Frequency Questionnaire to Assess Compliance to a Cardioprotective Mediterranean Diet

Background: The cardioprotective effects of the Mediterranean diet have been well established in epidemiological studies. More recently, it has been shown to improve the cardiometabolic risk profile and endothelial function, as well as reduce markers of vascular inflammation, independent of weight loss. Since cardiovascular disease is the second cause of death in the Canadian and North American populations, randomized clinical trials evaluating the efficacy of the Mediterranean diet in high-risk primary and secondary prevention are warranted. In order to conduct such studies, validated dietary assessment methods specific to population and food habits are needed. Therefore, the aim of our study was to assess the reproducibility and the relative validity of a quantitative food frequency questionnaire (FFQ) to be used in clinical trials at the Montreal Heart Institute’s Prevention Center. Methods: Fifty (50) participants (54% (27 of 50) women) aged 19 to 86 years with and without coronary disease were recruited, and randomized in 3 groups in a crossover design where the sequence of administration of questionnaires differed in each group. The FFQ includes 157 food items and was designed to measure food intake over one month. It was administered twice 3 to 5 weeks apart to assess reproducibility and was compared to a 12-day dietary record carried out over a 1-month period to assess validity. Participants were asked not to modify their diet for the duration of the study. FFQs were self-administered and reviewed by a registered dietician. All questionnaires were analyzed using The Food Processor software. Results: Reproducibility (n = 49) assessed by the intraclass correlation coefficient (ICC) revealed good (ICC &gt; 0.5) to very good (ICC &gt; 0.7) correlations for energy and all nutrients (except vitamin A) with ICCs ranging from 0.48 (95% confidence interval (CI), 0.23 to 0.67) for vitamin A to 0.91 (95% CI, 0.85 to 0.95) for alcohol (mean 0.68). Preliminary results for validity (n = 15) revealed very good significant correlations for EPA, DHA, selenium and alcohol and good significant correlation for energy, total fat, saturated fatty acids, monounsaturated fatty acids, oleic acid, linolenic acid, trans fat, vitamin B3, vitamin D, iron and magnesium. Conclusions: Our FFQ demonstrates good to very good reproducibility. Furthermore, the FFQ appears valid for evaluating adherence to a Mediterranean diet, notably because it provides very good estimates of marine omega-3 fatty acid (EPA, DHA) intake. These results support the eventual use of our FFQ in clinical trials on the Mediterranean diet. Complete sample analysis will be available in March 2014.

Rapid Column Extraction Method for Uranium in Urine

The conventional method used for estimation of uranium (U) in bioassay samples takes nearly 4–5 days for complete sample analysis. In cases of incidental exposures, there is a need to reduce overall analysis time for the estimation of U in bioassay samples. Therefore, efforts were made in the present study to develop a comparatively rapid method for analysis of U in urine. For this purpose, a known amount of U(natural) standard was added to synthetic urine samples (1 L) and separated from by solid phase extraction chromatography using TRU resin (Eichrom Technologies, Inc.). The radiochemical yield recovery observed for U was 92.6% by this method. As a part of quality assurance, the method standardized in the present study was applied for estimation of U in urine samples collected from members of the public (MOP) and the results were compared to those previously obtained using ion exchange chromatography and fission track analysis (FTA).

A method for estimation of Pu-isotopes in urine samples using TEVA resin and alpha spectrometry

The conventional method used for estimation of Pu-isotopes in urine samples involves anion exchange resin followed by alpha spectrometry, which takes nearly one working week for complete sample analysis. Since the results of the analysis form an important input for decision making by the plant authorities, it is always preferable to reduce overall analysis time for the estimation of Pu-isotopes in bioassay samples. This paper deals with standardization of a relatively faster method for estimation of Pu-isotopes in bioassay samples using TEVA resin and 236Pu tracer for radiochemical recovery. The method involves oxidation of urine followed by co-precipitation of plutonium along with calcium phosphate and separation of Pu was carried out using TEVA resin. Pu-fraction was electrodeposited and activity estimated using tracer recovery by alpha spectrometer. Routine urine samples of radiation workers were analyzed and consistent radiochemical tracer recovery was obtained in the range 65–87 % with a mean and SD of 75 and 7.4 %, respectively. The standardized chromatographic technique reduces the analysis time by about 1 day as compared to conventional method for estimation of Pu-isotopes in urine samples.

Preliminary results of a factorial phase II randomized trial of continuous (C) or intermittent (I) docetaxel (DOC) with or without estramustine (E) as first-line treatment for castration-resistant prostate cancer (CRPC) (HOPLITE trial).

220 Background: DOC given for 8 consecutive courses is considered a standard first line treatment for CRPC pts and I administration could reduce its impact on quality of life (QL). E is considered synergistic with DOC. Aim of this study was to evaluate in a 2 × 2 factorial design, if I DOC could improve QL compared to C DOC and whether E added to DOC could improve its activity. Methods: CRPC pts were randomized to: C DOC 70 mg/m2 i.v. q 3 wks for 8 courses, alone (arm A) or with E 280 mg/TID p.o. for 5 days starting on -1 day (arm B), or the same treatments given with a 3-month rest period after the first 4 courses (arm C and D, respectively). The primary end points were QL (EORTC QLQ C30 and BPI) of A+B vs C+D and 1-y PFS (according to PCWG2) of A+C vs B+D. Results: From 11/06 to 10/10, 148 CRPC pts were enrolled and 124 pts are presently evaluable (24 too early). The median age was 69 (range 42–81) and the median baseline PSA was 55.6 (range 0.33–4212). The major hematological toxicities were: anemia G3 (3 pts), neutropenia G3 (4 pts) – G4 (5 pts), febrile neutropenia (5 pts). QL outcomes of C and I groups, were not statistically different in terms of general QL items. 1-y PFS was also superimposable (10% and 13.5%, respectively) for DOC and DOC+E groups. The 2-y overall survival was also evaluated with no differences between I and C groups (42.5% and 53.7% respectively) and between DOC and DOC+E groups (42.8% and 53.5% respectively). Conclusions: These preliminary results seem to indicate that I treatment may not improve QL compared to C treatment. Moreover, the addition of E to DOC did not improve 1-y PFS of CRPC pts. Updated data with the complete sample analysis will be presented.

Validation of the Harmless Acute Pancreatitis Score in Predicting Nonsevere Course of Acute Pancreatitis

Background: The Harmless Acute Pancreatitis Score (HAPS) is a scoring algorithm to identify patients with nonsevere acute pancreatitis. The aim of this study was to evaluate the reproducibility of HAPS outside its original study setting. Method: Baseline information of all hospitalized patients with acute pancreatitis at Karolinska University Hospital, Stockholm, Sweden, between 2004 and 2009 was collected. The parameters constituting HAPS were signs of peritonitis, hematocrit and serum creatinine levels. Since hematocrit was not available in all patients, complete sample analysis was performed by replacing hematocrit with hemoglobin (strongly correlated with hematocrit; r = 0.86). Results: In total, 531 patients with a first-time or a recurrent attack of acute pancreatitis were included. Among 353 patients with complete information on parameters constituting HAPS, 79 patients were predicted to have a nonsevere course, of whom 1 patient developed severe acute pancreatitis. The specificity of HAPS in predicting a nonsevere course of acute pancreatitis was 96.3% (95% CI: 81.0–99.9) with a corresponding positive predictive value of 98.7% (95% CI: 93.1–100). Complete sample analysis replacing hematocrit with hemoglobin level predicted a nonsevere course in 182 patients, of whom 2 patients had severe acute pancreatitis (94.3% specificity and 98.9% positive predictive value). Conclusion: HAPS is a highly specific scoring algorithm that predicts a nonsevere course of acute pancreatitis. Therefore, HAPS might be an additional tool in the clinical assessment of acute pancreatitis where early screening is important to treat the patients at an optimal level of care.

Novel approach to calibration by the complementary dilution method with the use of a monosegmented sequential injection system

Novel approach to calibration by the complementary dilution method (CDM) is presented. The CDM integrates in a single procedure the set of standards and the standard addition calibration methods. The approach is implemented with the use of the fully automated monosegmented sequential injection system coupled to FAAS as a detector. It relies on generation of a series of standard solutions consistent with the CDM calibration method in subsequent monosegments using a single stock standard solution, sample and diluent in appropriate proportions. Two versions of the method, basic and extended have been verified. They have been tested on the example of magnesium determination in a synthetic sample with errors of repeatability (R.S.D.) and accuracy (R.E.) less than 3.2%. Subsequently, they have been applied to magnesium and calcium determinations in water. The results obtained are comparable with the appropriate certified values as well as with the results received by traditional calibration methods performed separately in batch system. Using the system, a complete sample analysis takes 8 or 21 min with sample or standard consumption less than 1 or 2 mL in basic and extended versions, respectively.

Four-Way Data Coupled to Parallel Factor Model Applied to Environmental Analysis: Determination of 2,3,7,8-Tetrachloro-dibenzo-para-dioxin in Highly Contaminated Waters by Solid−Liquid Extraction Laser-Excited Time-Resolved Shpol'skii Spectroscopy

This article reports the first application of parallel factor analysis to high-order instrumental data generated from Shpol'skii matrixes at liquid helium temperature. Third-order data arrays-consisting of excitation modulated wavelength time matrixes-are collected with the aid of a cryogenic fiber-optic probe, a tunable dye laser, and a multichannel system for phosphorescence detection. The multidimensional data formats are applied to the analysis of 2,3,7,8-tetrachloro-dibenzo-para-dioxin in water samples. The experimental procedure is rapid and environmentally friendly. Complete sample analysis is accomplished in less than 15 min with only 100 muL of organic solvent (n-heptane). The feasibility to directly determine parts-per-trillion concentration levels of the target compound is demonstrated with heavily contaminated samples of unknown composition. The limits of detection, estimated from calibrations based on the univariate method and parallel factor analysis, are 0.060 and 0.092 ng.mL(-1), respectively; both referred to a 100 mL water sample.

Rapid Quantitation of a Large Scope of Eicosanoids in Two Models of Inflammation: Development of an Electrospray and Tandem Mass Spectrometry Method and Application to Biological Studies

Assessment of eicosanoid levels in biological systems is important for understanding their role in cell function and pathophysiological events. Current methods of eicosanoid quantitation are limited by sensitivity, scope, or throughput. The development of a new method for eicosanoid assessment in biological samples by electrospray and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring mode is described here. In this study, 14 biologically significant eicosanoids were quantitated in a single sample. Complete sample analysis required two repeated injections of 5 μl with an analysis time of 1.5 min/injection. Limits of detection ranged from 0.5 pg for thromboxane B2(TxB2) to 10 pg for 6-keto prostaglandin F1α(6-keto PGF1α). The reliability, reproducibility, sensitivity, and cross-detection of the method is also described. The MS/MS method was used to explore eicosanoid production in two inflammation models: lipopolysaccharide (LPS)-stimulated human whole blood and carrageenan-challenged rat air pouch. The most abundant metabolites in LPS-stimulated whole blood were prostaglandin E2(PGE2), TxB2, and 6-keto PGF1α; prostaglandins E1, D2, and F2αand leukotrienes B4and C4were detected in lower amounts. Eicosanoid levels determined by MS/MS were similar to those obtained by immunoassay and GC-MS. The most abundant metabolites detected in carrageenan-challenged rat air pouch were PGE2, 6-keto PGF1α, and TxB2. The method described in this work is accurate and rapid and should greatly aid in evaluating the role of multiple eicosanoids in future biological studies.

Fast determination of elements in water, urine and serum by graphite furnace atomic absorption spectrometry

An alternative technique for sample drying is presented for graphite furnace atomic absorption spectrometry. The sample is dispensed to contact a pre-heated graphite tube immediately. The majority of the sample volume evaporates on contact with the graphite tube, greatly reducing the cycle time of sample analysis. Graphite furnace programme times of less than 20 s are achieved and a complete sample analysis, including sampling time, is achieved in less than 60 s. Methods are presented for the determination of chromium, copper and nickel in water, chromium and cadmium in urine and copper in serum. Relative standard deviations of absorbances are less than 4%. The accuracy of the technique is established from the analysis of reference materials.