Repeatability Precision Research Articles

Determination of Biogenic Amines in Different Parts of Lycium barbarum L. by HPLC with Precolumn Dansylation.

The aim of this work was to characterize biogenic amines (BAs) in different parts of Lycium barbarum L. using HPLC with dansyl chloride derivatization, and jointly, to provide referential data for further exploration and utilization of Lycium barbarum L. The linear correlation coefficients for all BAs were above 0.9989. The limits of detection and quantification were 0.015–0.075 and 0.05–0.25 μg/mL, respectively. The relative standard deviations for the intra-day and inter-day precision were 0.66–2.69% and 0.91–4.38%. The described method has good repeatability and intermediate precision for the quantitative determination of BAs in different parts of Lycium barbarum L. Satisfactory recovery for all amines was obtained (79.3–110.3%). The result showed that there were four kinds of BAs. The highest putrescine content (20.9 ± 3.2 mg/kg) was found in the flower. The highest histamine content (102.7 ± 5.8 mg/kg) was detected in the bark, and the highest spermidine (13.3 ± 1.6 mg/kg) and spermine (23.7 ± 2.0 mg/kg) contents were detected in the young leaves. The high histamine (HIS) content in the bark may be one of the reasons why all of the parts of Lycium barbarum L., except the bark, are used for medicine or food in China. Meanwhile, the issue of the high concentration of HIS should be considered when exploiting or utilizing the bark of Lycium barbarum L.

Optimized Chromatographic Determination of Naphazoline and Chlorpheniramine in Eye/Nose Drops Using Cation-exchange Column.

A simple and efficient liquid chromatographic method has been developed and validated for the simultaneous determination of naphazoline and chlorpheniramine in eye/nose drops, in presence of naphazoline degradation product and naphthalene acetic acid (NAPD). The separation was achieved within 6.0min, employing a mixture of 53.5% v/v water-acetonitrile containing 78.70mM/L sodium dihydrogen orthophosphate adjusted to pH5.30 as isocratic mobile phase, pumped at 1.0mL/min through a strong cation-exchange column (10μm particle size), the analytes were monitored at 230nm. Statistical experimental designs and graphic representations (response surface methodology) were used for optimizing the chromatographic separation. The linearity plots were linear over concentration range up to 125% of the analytes nominal concentrations (100%) with regression coefficients (r) > 0.99, method's accuracy (RSD < 2.0%), repeatability and intermediate precision (RSD < 2.0%) were verified. System suitability parameters were also within the acceptable range. The validated method was successfully employed for the routine analysis of eye/nose drops.

In-situ synthesis of nanocubic cobalt oxide @ graphene oxide nanocomposite reinforced hollow fiber-solid phase microextraction for enrichment of non-steroidal anti-inflammatory drugs from human urine prior to their quantification via high-performance liquid chromatography-ultraviolet detection

The in-situ synthesis and application of nanocubic Co3O4-coated graphene oxide (Co3O4@ GO) was introduced for the first time to present a cost-effective, stable and convenient operation and a simple device for hollow fiber solid-phase microextraction (HF-SPME) of four selected nonsteroidal anti-inflammatory drugs (NSAIDs) including diclofenac, mefenamic acid, ibuprofen and indomethacin. The extracted analytes were desorbed by an appropriate organic solvent and analyzed via high-performance liquid chromatography-ultraviolet detection (HPLC-UV). The prepared sorbent was approved using different characterization methods such as X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The variables effective on the Co3O4@GO-HF-SPME method including extraction time, desorption time, desorption solvent volume, sample pH, stirring rate and ionic strength were screened via Plackett–Burman design and then optimized by Box–Behnken design. Under optimal condition, the calibration curves were linear within the range of 1.0–200.0 µg L−1 of analyte concentration with detection limits of 0.18–1.1 µg L−1 and the relative standard deviations less than 10.1%. The limits of quantification (LOQs) were in the range of 0.60–3.67 µg L−1. Matrix effect was not observed with this method; therefore, standard addition is not necessary for quantification of target compounds. The enrichment factors were obtained in the range of 49–68. The relative recoveries of the urine sample analysis were calculated in the range of 93–102%. Finally, the presented method exhibited good sensitivity, excellent repeatability, high reusability and acceptable precision, which will be a promising method to analyze various nonsteroidal anti-inflammatory drugs in urine samples.

Development and Validation of an Automated Zone Fluidics-Based Sensor for In Vitro Dissolution Studies of Captopril Using Total Error Concept.

In the present research, a zone fluidics-based automated sensor for the analysis of captopril in in vitro dissolution samples is reported. Captopril is reacted under flow conditions with Ni(II) (10 mmol L−1) in alkaline medium (0.15% v/v NH3) to form a stable derivate, which is monitored spectrophotometrically at 340 nm. The chemical and instrumental parameters were carefully investigated and optimized. The validation of the developed method was performed in the range of 5 to 120% of the expected maximum concentration using the accuracy profiles as a graphical decision-making tool. The β-expectation tolerance intervals did not exceed the acceptance criteria of ±10%, which means that 95% of future results will be encompassed in the defined bias limits. The variation of the relative bias ranged between −2.3% and 3.5% and the RSD values for repeatability and intermediate precision were lower than 2.3% in all cases. The limit of detection (LOD), and the lower and the upper limit of quantification (LLOQ, ULOQ) were satisfactory and found to be 1%, 5% and 120% (corresponding to 0.6, 2.78 and 66.67 μg mL−1 in dissolution medium). The developed method was successfully applied for the analysis of captopril in dissolution tests of two commercially available batches.

Development and validation of UPLC method for quantitative determination of major bovine milk oligosaccharides and their heat stability

Bovine milk oligosaccharides (BMOs) and human milk oligosaccharides (HMOs) have similar functions. In this investigation, a simple and rapid quantitative analysis method of UPLC for BMOs concentration was proposed, and the heat stability of BMOs was also revealed. BMOs were separated on an Acquity UPLC® Glycan BEH amide column (2.1 mm × 150 mm, 1.7 μm) using 100 mm ammonium formate (pH 4.5) and 100% acetonitrile as mobile phase for gradient elution at a flow rate of 0.45 mL min−1. The potential in quantification of BMOs were satisfactorily validated in terms of linearity (R2 ≥ 0.9992), repeatability and intermediate precision (RSD ≤ 4.46%), and spike recovery (≥90.27%). For all investigated oligosaccharides, LOD ranged from 1.97 to 9.38 μg L−1 and LOQ values ranged between 6.84 and 31.25 μg L−1. In addition, BMOs did not degrade after heating at 45–100 °C for 10–120 min.

A Study on the Test Device for Improving Test Speed and Repeat Precision of Semiconductor Test Socket

A Study on the Test Device for Improving Test Speed and Repeat Precision of Semiconductor Test Socket

Separation and Rectification of Chloroacetyl Chloride from TiCl4

Titanium tetrachloride (TiCl4) is an important intermediate material for the preparation of titanium products. The organic impurities in TiCl4 are easily accumulated during the production of titanium sponges due to the problems of imperfect detection methods and the lack of effective control methods, resulting in a poor quality of sponge titanium. Among all impurities, chloroacetyl chloride (CAC) is the most important in TiCl4. Herein, the determination of the CAC content in TiCl4 solution, with a low detection limit of 0.633 ppm, was established by the standard addition method using Fourier transform infrared (FTIR) spectrometry. This test method presented good repeatability, excellent accuracy, and moderate precision. Furthermore, the influencing factors of CAC separation in the continuous rectification process, including the heating power (the ratio of total heating power to feed rate), reflux temperature, top tower pressure, and feed temperature were optimized based on an orthogonal experimental design. The experimental data demonstrated that the average CAC removal rate reached 78.94% ± 1.00% under the optimal distillation conditions, with 72.21% of the CAC removed via the off-gas system. Therefore, excellent control of the negative pressure of the tail gas is highly desirable for the removal of CAC impurities.

Dosage of sodium and potassium in paediatric parenteral nutrition bags: Using an automated system for physicochemical control

Summary Introduction Validate an analytical method for the determination of Sodium and Potassium in parenteral nutrition bags by indirect potentiometry using correction factors; and evaluate routine control results. Material and methods An analytical method of dosage of electrolytes by Cobas Integra 400 Plus® analyzer was validated using the SFSTP guidelines. Bags of matrix admixtures were prepared for validation; electrolytes were added at different concentrations. We studied the linearity, accuracy and precision. The validated method was used to assess the conformity of electrolytes concentrations within the parenteral nutrition bags. The acceptance limit for conformity assessment was set at ± 20% of the target concentration. Results Correlation coefficients of the regression lines were higher than 0.999. The method showed good repeatability and intermediate precision (RSD Discussion Non-conformity is increased by increasing the total volume and exceeding the theoretical capacity of the bag. The extension of the mixing time significantly increases the conformity rate. Conclusion A laboratory analyzer provides a valid and precise method for the determination of electrolytes in parenteral nutrition bags. A proper mixing is critical in order to obtain exact determination results.

Modified QuEChERS/UPLC-MS/MS method to monitor triclosan, ibuprofen, and diclofenac in fish Pseudoplatystoma magdaleniatum

The objective of the study was to develop a simple, fast, and sensitive analytical method for the extraction and quantification of ibuprofen, diclofenac, and triclosan in the muscle of the striped catfish Magdalena (Pseudoplatystoma magdaleniatum), a species of fish emblematic of the main rivers of Colombia (Magdalena and Cauca) that is critically endangered. The extraction method used was QuEChERS, the cleaning method for the removal of co-extracted fats and lipids was freezing, and quantification was performed by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The method was validated according to the performance requirements of Appendix F of AOAC. The results showed that the method is accurate and reproducible; the extraction procedure is fast and can be performed on a small-sample aliquot. The value of LOQ in fish tissue was 20 μg/kg dry weight and LOD of 2.775 for diclofenac; 1.163 for ibuprofen; and 1.214 for triclosan. RSDs for repeatability and intermediate precision were < 20.3%. Linearity has correlation coefficients (r) greater than 0.99 for the range of 20–400 μg/kg. Recoveries of 63–112%, 75–102%, and 62–113% for triclosan, diclofenac, and ibuprofen, respectively, were obtained. The method was applied to fish in the Magdalena and Cauca rivers with positive results, finding the polluting triclosan in the fish muscle at concentrations of up to 54.67 μg/kg dry weight, while ibuprofen and diclofenac were not detected in the matrices analyzed. This is the first freshwater fish research in Colombia.

A green molecular imprinted solid-phase extraction protocol for bisphenol A monitoring with HPLC-UV to guarantee the quality and safety of walnuts under different storage conditions.

Monitoring the residual toxicant concentrations in foods is the key step for minimizing potential hazards. The huge interest about food contamination and exposure to endocrine disruptors such as bisphenol A has emerged the development of sensitive analytical methodologies to guarantee the safety and quality of foods. In this work, a green molecularly imprinted solid-phase extraction protocol coupled with high-performance liquid chromatography with UV detection was optimized following the principles of green analytical chemistry. An imprinted sol-gel silica-based hybrid inorganic-organic polymeric sorbent was used to monitor the leaching of bisphenol A from different packaging materials (glass vessels, cans, and polypropylene containers) in walnuts stored within a period of 6 months at 25 and 4°C. Extraction parameters including loading time (5-20min), solvent type (acetonitrile, ethanol, methanol, acetone, acetonitrile:methanol, 50:50, v/v), and elution flow rate (0.2-1mL/min) were optimized with one-factor-at-a-time method. The selected extraction optimum parameters incorporated elution with acetonitrile at 0.2mL/min flow rate, for 10min sample holding time. The imprinting factor was equal to 4.55 ± 0.26 (n=3). The optimized method presented high recovery (94.3 ± 4.2%, n=3), good linearity(>0.999), intra-assay repeatability (90.2-95.6%, n=3), and interassay precision (86.7-93.1%, n=3).

Development of Analytical Method for Determination of p-toluenesulfonylurea and its Impurities in Gliclazide Drug Substances by using HPLC

A highly sensitive analytical method is developed to determine the p-toluenesulfonylurea (PTSU) and its impurities namely o-toluenesulfonamide (OTSA), p-toluenesulfonamide (PTSA) and p-toluenesulfonic acid (PTS) in Gliclazide drug substance by high performance liquid chromatography (HPLC). The developed method is validated according to the International Council for Harmonization (ICH) guidelines. The method is specific where peak purity is passing for all the impurities and were well separated. Limit of detection and limit of quantitation established for all the four impurities which is less 30% of target concentration of 0.1%. The regression coefficient found from linearity studies was greater than 0.9922 for all the four impurities. The % recovery obtained for all the impurities in Gliclazide was between 86.56% and 105.21%. Precision, repeatability and intermediate precision are established for the method by observing the relative standard deviation (RSD) which is less than 10%. The developed method can be adapted by quality control to determine and quantify these impurities in Gliclazide drug substance or drug product.

A validated Folin-Ciocalteu method for total phenolics quantification of condensed tannin-rich açaí (Euterpe oleracea Mart.) seeds extract

The widely used methodology to quantify polyphenols-the Folin-Ciocalteu (FC) method-cannot be applied indiscriminately since different matrices may impair the assay's accuracy. Thus, this study aimed to adapt the FC method for the açaí seed extract, a tannin-rich extract with potential applications for various therapies. Firstly, a pre-method standardization was established to determine parameters such as reading wavelength (765nm), reaction time (30min), and the reference substance (pyrogallol). In the validation step, the adapted method responded linearly to the analyte (R2 = 0.9910), ensuring its selectivity (linearity and selectivity curves statistically parallel) and accuracy (99.18-101.43%). Furthermore, the method proved to be precise (RSD ≤ 2.63%) at the two levels assessed (repeatability and intermediate precision) and robust (RSD ≤ 4.45%) concerning variation on the Na2CO3 concentration and the reaction time. The limits of detection and quantification were also calculated (9.9µg/mL and 33.1µg/mL, respectively). An additional step for tannins quantification based on its reported selective precipitation by complexing agents was also evaluated; however, unspecific precipitation was observed, reducing the results' accuracy. Our work successfully adapted and validated a method for total phenolics quantification of açaí seed extract, resulting in 38g of pyrogallol equivalent/100g of extract.

Method development and validation for the determination of residual solvents in quinabut API by using gas chromatography. Message 2

Aim. The aim of study was to develop and validate a simple, precise and accurate method using gas chromatography for analysis of residual solvents – acetone and 2-propanol – in quinabut API. Materials and methods. All experiments were performed on a gas chromatographic system equipped with FID detector (Shimadzu GC System) using the DB-624 (30 m × 0.32 mm ID, 3.0 μm film sickness) column as stationary phase. Nitrogen was used as carrier gas with flow rate 7.5 mL/ min. Split ratio was 1:5, injector temperature was 140 °C, detector temperature was 250 °C, oven temperature was programmed from 40 °C (2 min) to 50 °C at 1 °C/min and then increased at a rate of 15 °C/min up to 215 °C; and maintained for 2 min. All solutions were prepared using water as diluent. Results. This proposed method is assessed for separation of residual solvent from quinabut with quantification. The obtained results are compared with the corresponding specified limits of ICH standard guidelines. The method validation was done by evaluating specificity, limit of detection (LOD) and limit of quantitation (LOQ), linearity, accuracy, repeatability, ruggedness, system suitability and method precision of residual solvents as indicated in the ICH harmonized tripartite guideline. The separation between acetone and 2-propanol peaks is 2.07. Hence method was found to be specific. The linear relationship evaluated across range of 15 to 180% for acetone and 2-propanol of ICH specified limit of residual solvents. The graphs of theoretical concentration versus obtained concentration are linear and the regression coefficients ‘R’ for residual solvents were more than 0.9968. The values of LOD and LOQ were much less than the lower limit of the concentration range and cannot affect the accuracy of the test. The technique was characterized by high intra-laboratory accuracy at concentrations close to the nominal acetone and 2-propanol concentration. All solutions were stable in water for at least 1 hour when stored at room temperature. Conclusion. A simple, specific, accurate, precise and rugged gas chromatography method was developed and validated for the quantification of residual solvents present in quinabut API through an understanding of the synthetic process, nature of solvents and nature of stationary phases of columns. The residual solvents acetone and 2-propanol were determined.

A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates

We present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

Diagrammatic scales for the estimation of black node disease severity in common bean

ABSTRACT: The objective of this study was to design two diagrammatic scales for the evaluation of black node disease (Stagonosporopsis hortensis and Boeremia spp.) in common bean (Phaseolus vulgaris L.). The developed scales corresponded to logarithmic intervals (SADL) of seven levels and equal intervals (SADE) of nine levels. These scales were compared to the scale developed by the International Center for Tropical Agriculture (CIAT), which corresponds to a descriptive scale (NoSAD). In the scale validation the accuracy, precision, and concordance of repeatability were determined using Lin’s concordance correlation coefficient, whereas inter-rater reproducibility was determined by the overall concordance correlation coefficient (OCCC). It was observed that with the use of the proposed scales, reproducibility and repeatability were significantly improved for inexperienced raters, and reproducibility was improved in the case of experienced raters. Thus, the designed standard area diagrams with equal and logarithmic intervals are a useful tool for estimating severity under field and experimental conditions as part of the study of this patho-system.

Development and validation of RP-HPLC method for simultaneous determination of lamivudine, stavudine, and zidovudine in perfusate samples: Application to the Single-Pass Intestinal Perfusion (SPIP) studies

A reversed-phase high performance liquid chromatography (RP-HPLC) method with ultraviolet detection was developed and validated for the simultaneous quantification of antiretroviral drugs lamivudine (3TC), stavudine (d4T), and zidovudine (AZT) in perfusate samples obtained from the Single-Pass Intestinal Perfusion studies. The chromatographic analysis was performed using a Gemini C18 column and didanosine as internal standard (IS). The following parameters were considered for the validation procedure: system suitability, accuracy, precision, linearity and selectivity. The limits of detection were 0.32 µg/mL for 3TC, 0.11 µg/mL for d4T and 0.45 µg/mL for AZT and the limits of quantification were 1.06 µg/mL for 3TC, 0.38 µg/mL for d4T and 1.51 µg/mL for AZT. Repeatability and intermediate precision ranged from 1.05 to 1.31 and 1.50 to 1.87, respectively, and are expressed as percent of relative standard deviation (RSD). Based on these results, the developed and validated RP-HPLC method can be used for simultaneous determination of 3TC, d4T, and AZT in perfusate samples. Furthermore, this method is simple and adequate for measurements of the antiretroviral drugs in the same sample, since those compounds are mostly co-administered. Besides, this work can be used as an initial base for the development of similar methods in the same conditions presented in our study.

Development and validation of an UV-Vis spectrophotometric method for the quantification of oclacitinib in capsule formulation

Objective. The aim of this study was the development and validation of an UV-Vis spectrophotometric method for the quantification of oclacitinib in commercial capsule formulation since pharmacopeias have not yet provided an official monograph for this drug. Methods. The parameters linearity, limit of detection, limit of quantitation, specificity, precision, accuracy, and robustness were determined according to Brazilian and international guidelines. Results. Linearity was determined for the analytical range of 5-15 μg/mL, and a limit of detection of 1.18 μg/mL and limit of quantification of 3.58 μg/mL were obtained. The method was selective and the precision was demonstrated through repeatability and intermediate precision, with relative standard deviations of 1.96% and 1.78%, respectively. In its turn, accuracy presented recovery percentages of 98.32-100.91%. All robustness and sample stability (48 h at 25 °C) results revealed no statistical variation among the groups. Conclusions. The presented method is suitable for the quantification of oclacitinib in commercial capsule formulation.

Analytical Evaluation of Lumipulse Anti-Mullerian Hormone Assay.

The measurement of serum Anti-Mullerian hormone (AMH) is used in daily practice to estimate the ovarian reserve in women. The aim of this study was to evaluate the new Lumipulse AMH® immunoassay (Fujirebio) with regard to the reliability with two preexisting assays: Elecsys AMH Plus® (cobas®, Roche) and Vidas AMH® (bioMérieux). Precision of Lumipulse AMH was evaluated on the Lumipulse G600 II using the manufacturer's quality controls. Thirty-three samples were used for method comparison. Lumipulse AMH repeatability and intermediate precision did not exceed 4.1 %. There was a proportional bias between Lumipulse and Cobas method and between Lumipulse and Vidas whereas a good agreement was found between Cobas and Vidas. Lumipulse AMH assay demonstrated good precision and good agreement with the two other methods up to 6 ng/mL. Nevertheless, this study shows the need to standardize AMH assays to improve the comparability of the methods.

Development of a TD-NMR Method to Monitor Brazil Nuts Oil Content: A Green and Low-Cost Based Approach

Growth in the international popularity of Brazil nuts oil (Bertholletia excelsa, Bonpl.) has demanded a closer look at quality aspects of the nuts. In this context, time-domain 1H nuclear magnetic resonance spectroscopy (TD-NMR) can be a useful tool for assessing the lipid portion of such matrices. In this work, a TD-NMR method was developed and validated to assess the oil content directly from Brazil nuts following the ISO 10565:1998 and DOQ-CGCRE-008. Full validation was addressed covering the working range from 0.67 to 25.00 g (coefficient of determination, R2 = 0.9993). Limits of detection (LOD) and quantification (LOQ) were 0.20 and 0.67 g, respectively. The repeatability and intermediate precision assays met the guide requirements. Oil contents obtained in two laboratories (different equipment) were statistically similar (66.93 ± 4.69 and 70.13 ± 4.15%), highlighting the method’s robustness. The developed approach has the potential to be adopted as an industrial reference method for in situ monitoring of Brazil nuts oil contents.

Determination of sweeteners in wine by liquid chromatography coupled with mass spectrometry (LC/MS)

Sweeteners are food additive substances that give a sweet taste to foods but their use in oenological practices is forbidden. Making use of the capabilities of liquid chromatography coupled with mass spectrometry, a method for wine analysis was developed and validated for the detection and quantitation of some of the most widely used sweeteners: aspartame, potassium acesulfame, sodium cyclamate, saccharin, sucralose and stevioside. A matrix-matched calibration was used for all compounds obtaining a linear concentration range from 50 μg/L to 1000 μg/L. The limit of detection ranged from 0.002 mg/L to 0.014 mg/L, and the limit of quantification varied between 0.005 mg/L and 0.048 mg/L. Precision and recovery were assessed for 50 μg/L, 250 μg/L and 1000 μg/L with repeatability and intermediate precision values from 0.6% to 21.6% and 2.7% to 26.4% respectively, and recoveries ranging from 60% to 126%. These results were achieved using minimal sample preparation with a fast and high throughput method that is applicable to a wide range of wine matrices.