Spectrometer configuration and measurement uncertainty in X‐ray spectroscopy

Although reluctant to monopolize debate on the meaning of “sensitivity”, we feel compelled to respond to Dr. Pardue’s critique (1), which reveals certain misunderstandings and misrepresents our arguments (2). Pardue claims that the “formal definition of sensitivity” is associated with the response/stimulus ratio. But formal definitions have differed, certain authorities defining sensitivity in terms of the detection limit. Elimination of the contradictions underlying the scientific use of the word has been our objective in raising this issue. Contrary to Pardue’s belief (1), we do not argue that the “slope interpretation conflicts with the formal definition”. We comprehend the equivalence of the “slope” and “response/stimulus” definitions, but object to both on the grounds that they conflict with the fundamental meaning of sensitivity. We maintain, in short, that neither is a valid indicator of the ability of an instrument to sense small changes “in that to which it is designed to respond”, and hence of its sensitivity in the generally accepted sense. We nevertheless agree that the response curve slope may be “an invaluable descriptor of one of the most important characteristics of any analytical method” (1), albeit only when combined with estimates of the statistical uncertainty in response measurements. Lacking these, it is impossible to determine whether an observed change in the response constitutes a random fluctuation or reflects a real difference in the measured quantity. In short, the response curve slope is simply a property of the curve (as plotted), not a meaningful indicator of the analytical performance of a system. Pardue claims that “proper use of the slope definition yields much more information than is available in the ’detection limit’ interpretation” (1). However, his view of “proper use” entails combining the slope with “the uncertainty in the measurement response” (termed er). This yields the “quantitative resolution” …

Objective: Nitrosamine impurities, particularly nitrosamine drug substance-related impurities (NDSRIs), have gained focus in recent past due to their carcinogenic potential. Due to the low sensitivity of conventional techniques like high-performance liquid chromatography (HPLC) and gas chromatography (GC), the exact levels of these impurities could not be determined. This study aims to develop and validate a sensitive liquid chromatography hyphenated with mass spectrometry (LC-MS/MS) method to estimate the trace levels of N-Nitroso fluoxetine (a fluoxetine-related NDSRI) in fluoxetine capsules (60 mg). Methods: We developed a sensitive and selective LC-MS/MS method for the quantification of trace levels of N-Nitroso fluoxetine in fluoxetine capsules (60 mg) using the AB Sciex 5500+quadrupole ion trap (QTRAP) LC-MS/MS system. To achieve the maximum response at quantitation level for N-Nitroso fluoxetine impurity, chromatographic conditions optimized by selecting a specific combination of column, mobile phase, and diluent. Mobile Phase A and B consisted of 10 mmol ammonium acetate buffer and methanol, respectively. Waters X-Bridge C18 column (250 × 4.6 mm, 3.5 μm, Part No. 186003943) at temperature 40 °C and mobile phase flow rate to 0.5 ml/min has yielded consistent results by employing an optimized gradient elution program (Tmin/%B): 0/75, 2/75, 16/95, 28/98, 28.1/75, and 35/75 for the analysis. To further maximize the response of the N-Nitroso fluoxetine impurity, the multiple ion reaction monitoring (MRM) mass spectrometry method in positive electron spray ionization (ESI) mode is preferred over single ion monitoring (SIM) method. Results: The newly developed method is validated by performing parameters such as specificity, method precision, intermediate precision, detection limit, quantitation limit, linearity, and accuracy in accordance with the international conference on harmonization (ICH Q2 (R1)) Guidelines. Limit of detection (LOD) and limit of quantitation (LOQ) are established as 0.06 ppm and 0.11 ppm, respectively. The method is found capable to demonstrate accuracy, precision, and linearity across the range of 0.11 to 1.68 ppm (10% to 150% of the specification), where linearity study shows the correlation coefficient (r²) greater than 0.99 for linearity plot between concentration and area. Recovery data at 10%, 50%, 100%, and 150% of the specification levels indicates that the recovered impurity content observed between 80% and 120%. Based on these results, we have successfully established the method for batch analysis of fluoxetine capsules (60 mg). Conclusion: A new rapid and sensitive reverse-phase ultra-high-performance liquid chromatographic (UHPLC) method coupled with a mass spectrometry detector has been developed and validated for the estimation of N-Nitroso fluoxetine impurity in fluoxetine capsules. Method validation is accomplished according to the ICH Q2 (R1) guidelines, assessing parameters such as limit of detection, limit of quantitation, accuracy, precision, and linearity. We found the method suitable for its intended use and successfully applied it to test batches of the fluoxetine capsules drug product.

The estimated global hard cider market is over $10 billion. Methods for determining their sugar content would have important implications in their quality assurance and nutritional values. In this study, the concentrations of fructose, glucose, and sucrose were determined in commercial hard cider and apple juice samples by hydrophilic interaction liquid chromatography with tandem mass spectrometry. The optimized sample preparation method consisted of sonication, a 1:10 dilution to 70:30 acetonitrile:water, centrifugation, filtration, and an additional 1:100 dilution to a final solvent composition of 80:20 acetonitrile:water. This method was validated with respect to selectivity, linearity, limit of detection, limit of quantitation, accuracy, and precision. Limits of detection and quantitation ranged from 0.01 to 0.1 and 0.05 to 0.5 mg/L, respectively, and linear ranges spanned at least a factor of 500 with excellent linearities ( R 2 > 0.999). Accuracy ranged from 81.7 to 107%. The relative standard deviations for all measurements were <7%. The method was applied to 11 different cider samples, one apple ale sample, and three different apple juice samples. Among the cider samples, a correlation between the fructose and glucose concentrations was found ( R 2 = 0.81).

Apigenin is one of the flavonoid compounds contained in celery ( Apium graveolens L.) and has been used for the treatment of various diseases. This study aims were to ensure the validity and accuracy of analytical method for apigenin assay in celery extract using UV-Vis spectrophotometry. The flavonoids test of celery extract give a positive result which produces a yellow color that can be drawn by amyl alcohol. The result of TLC test using a mobile phase of chloroform and methanol (9.5 : 0.5) produced R f value of 0.75. Validation of analytical methods were sensitivity, linearity, limit of detection as well as limit of quantification, accuracy, and precision. Validation of analytical methods showed that sensitivity results as represented by molar extinction (e) was 77004.35 M -1 cm -1 . Linearity from calibration curve at concentration of 2, 4, 6, 8, and 10 mgL -1 resulted an equation of y = 0.074 x – 0.0011 with coefficient correlation (r) of 0.9996. The limit of detection and limit of quantification were 2.99 mgL -1 and 9.92 mgL -1 respectively with recovery percentage of 97.59% and the coefficient variation values were 1.53%; 2.43% and 1.68%. The average contents of apigenin in the celery extract were of 3.74 ± 0.09%.

A new, simple, specific, and rapid ultra high performance liquid chromatography method was developed and validated for the determination of aripiprazole and its active metabolite dehydroaripiprazole in rat plasma. The analysis was performed on a Zorbax Eclipse Plus C18 (50 mm × 2.1 mm, 1.8 µm particles) column using gradient elution with a mobile phase consisting of acetonitrile and acetate buffer (30 mM, pH 5). Plasma samples were analyzed after a simple, one-step protein precipitation with acetonitrile. The method was validated and the specificity, linearity, limit of detection, limit of quantitation, precision, accuracy, recoveries, matrix effect, stability and robustness were determined. The analytes and internal standard were separated in 3.5 min and the total analysis time including the clean-up step was 8 min. Limit of detection was 0.012 µg mL -1 and 0.009 µg mL for aripiprazole and dehydroaripiprazole, respectively. Limit of quantitation was 0.039 µg mL -1 and 0.029 µg mL for aripiprazole and dehydroaripiprazole, respectively.The intra- and inter-day assay variability was less than 2% for the analytes. The proposed method is rapid, reproducible and accurate to quantify both aripiprazole and dehydroaripiprazole. It involves a simple plasma deproteination technique using one step protein precipitation with acetonitrile. This validated method was successfully used to quantify plasma concentrations of the analytes in rat plasma.

Highly sensitive spectrofluorimetric method was developed and validated for the determination of gabapentin in pure and pharmaceutical preparations. The method was based on nucleophilic substitution reaction of gabapentin with 4‐fluoro‐7‐nitrobenzofurazan (NBD‐F) in an alkaline medium (pH 9.5) to form a highly fluorescent derivative that was measured at 521 nm after excitation at 458 nm. The factors affecting the reaction was carefully studied and optimized. The method was successfully validated for linearity, limit of detection, limit of quantification, accuracy, precision, robustness and specificity. Under the optimized conditions, linear relationship with good correlation coefficient (0.9998) was found between the relative fluorescence intensity and gabapentin concentration in the range of 10–100 ng·mL−1. The limit of detection and limit of quantification were 0.43 and 1.30 ng·mL−1, respectively. The proposed method was successfully applied to the determination of gabapentin in its pharmaceutical capsules. The results obtained by the proposed method were comparable with those obtained by the official method. Statistical comparison by t‐ and F‐ tests revealed that there was no significant difference between the results of the two methods with respect to mean values and standard deviations at the 95% confidence level.

Atopic dermatitis is a typical chronic inflammatory skin disease that affects all age groups and requires basic skin care for treatment. Anti-inflammatory and antiallergy steroids are the most frequently used treatments but they are limited due to their side effects caused by a weakening of the immune system. Many consumers focus on performance as a criterion for selecting cosmetics. However, steroids have been illegally used to improve the performance of cosmetics, and consumers have been adversely affected by the corresponding side effects. In this paper, we propose a simple and rapid method using liquid chromatography-tandem mass spectrometry to simultaneously analyze ten non-permitted atopic therapeutic compounds in cosmetic products: chlorpheniramine maleate, ketotifen fumarate, doxepin hydrochloride, azelastine hydrochloride, bufexamac, clotrimazole, tranilast, fusidic acid, tacrolimus, and pimecrolimus. Additionally, the major characteristic fragment ions for tacrolimus, pimecrolimus, and clotrimazole were identified by time-of-flight mass spectrometry. The specificity, linearity, limit of detection, limit of quantification, recovery, precision, accuracy, and stability of the proposed method were validated. The limit of detection and quantification were in the ranges of 5.05-203.30 pg/mL and 15.15-609.90 pg/mL, respectively. The proposed analysis method could help improve the safety management of cosmetics.

BackgroundTumor marker carbohydrate antigen 15‐3 (CA15‐3) is used as a biomarker to aid to diagnose and monitor the prognosis of breast cancer patients. A new quantitative determination kit for CA15‐3 with chemiluminescent assay was developed by Xiamen InnoDx Biotech Co., Ltd, China. Therefore, we conducted the report to evaluate the performance of the kit.MethodsAccording to the “Guiding principles on performance analysis of diagnostic reagents in vitro”, the calibration curve, limit of detection, reportable range, accuracy, precision, anti‐interference capability, cross‐reaction and comparison by measuring EDTA plasma and serum were carried out. In addition, the kit was performed in parallel to electrochemiluminescence immunoassay kit (Roche) to analyze the correlation between the two kits.ResultsRegression equation of calibration curve of the kit was Y=0.7914X+4.1032 (R2=.990). Limit of detection was 0.0347 U/mL. The reportable range was 0.5‐2400 U/mL. Recovery ratio was 100.0%‐104.8%. Coefficient of variations (CVs) of within‐run and between‐run were 4.8%‐7.6% and 5.8%‐7.4% respectively. No remarkable interferences (all Bias% were less than ±10%) were detected when samples contained hemoglobin ≤183.8 μmol/L, bilirubin ≤340 μmol/L, triglyceride ≤18.1 mmol/L, or rheumatoid factor ≤400 U/mL. No cross‐reaction was present in the kit. Moreover, compared with the results from electrochemiluminescence immunoassay kit (Roche) in 345 serum samples, there was a satisfied correlation coefficient of 0.977 (P<.01), and the kit was simultaneously fit for the detection of EDTA plasma and serum samples.ConclusionThe new kit validated satisfactorily, and it can be used for detecting CA15‐3 in clinical practice.

Method validation of sodium dodecylbenzene sulfonate (SDBS) analysis in catfish by maceration and UV-Vis spectrophotometry using methylene blue active substances (MBAS) method was performed. The first stage of this research was method validation comprising correlation coefficient, sensitivity, Limit of Detection (LOD), Limit of Quantitation (LOQ), precision, and accuracy. The extraction of SDBS by maceration with methanol for 16 h and analysis by UV-Vis spectrophotometry using MBAS were performed. Then, this method was applied to analyze SDBS in catfish. The results of validation of SDBS analysis in cultivated catfish give correlation coefficient 0.9944, sensitivity 10.57 × 104 L mol-1 cm-1, LOD 0.01776 mg/L, LOQ 0.05922 mg/L, precision 0.1351-1.300 %, and accuracy 87.64-90.01 %, respectively. The SDBS concentrations in fish increase with the increase in the SDBS concentration in cultivating water. Bioconcentration Factor (BCF) values were inversely proportional to the concentration of SDBS in the cultivating water. The concentrations of SDBS on catfish, tilapia, and carp in traditional markets were 5.719-8.162; 5.180-6.548; and 2.973-5.874 mg/kg, respectively. In general, analysis method of SDBS in catfish by maceration and UV-Vis spectrophotometry with MBAS show a good result and can be used as a routine analysis in fish.

BackgroundPulmonary nocardiosis is an infection targeting immunocompromised patients characterized by high mortality and requires nonfrontline antibiotics for treatment. Nocardiosis is currently confirmed or excluded by BAL fluid culture followed by further phenotypic identification steps. A culture-independent method with more timely results would accelerate the administration of appropriate treatment. A rapid Nocardia (NOC) PCR assay for BAL has neither been previously validated nor offered for clinical testing to our knowledge.MethodsOligonucleotides for a rapid NOC PCR comprehensive of the causative agents of nocardiosis were aligned to the 16S regions of common NOC species and over 30 others. Specificity was verified against publically available bacterial 16S sequences. Rapid automated nucleic acid extraction (<1 hour for 24 samples) followed by fast PCR (<1 hour) was validated according to relevant compliance standards. Spiked/unspiked human BAL samples were used to assess analytical specificity, limit of detection (LOD), precision and accuracy using NOC and non-NOC strains.ResultsThe NOC PCR detected, among others, the most common NOC species (N. cyriacigeorgica, N. nova, N. farcinica and N. brasiliensis). We estimate more than 95% of causative agents of nocardiosis are detectable by the assay. No cross reactivity was detected from 30 non-NOC bacterial pathogens except for Rhodococcus and Crossiella spp. LOD in BAL fluid was determined to be 206, 41, and 26 copies/mL for N. cyriacigeorgica, N. nova, and N. transvalensis, respectively. Intra- and inter-assay precision studies revealed copies/mL %CV’s of <10% and <8% at a high concentration and <21% and <26% at a low concentration, respectively. Accuracy studies yielded 100% concordance with 33 BAL positives and 20 BAL negatives.ConclusionThe specificity, inclusivity, sensitivity, precision and accuracy of a qualitative PCR have been deployed as an aid in the diagnosis of pulmonary nocardiosis. NOC PCR allows for a culture-independent method that can rapidly detect clinically relevant NOC species with an improved turnaround time, leading to prompt diagnosis and administration of appropriate treatment.Disclosures E. Smith, Viracor Eurofins Clinical Diagnostics: Employee, Salary. K. Bartlett, Viracor Eurofins Clinical Diagnostics: Employee, Salary. J. Grantham, Viracor Eurofins Clinical Diagnostics: Employee, Salary. N. Powell, 1987: Employee, Salary. M. Altrich, Viracor Eurofins Clinical Diagnostics: Employee, Salary. S. Kleiboeker, Viracor Eurofins Clinical Diagnostics: Employee, Salary. M. Wissel, Viracor Eurofins Clinical Diagnostics: Employee, Salary.

Rifampicin (RIF) is the main treatment for tuberculous meningitis (TBM), but its limited penetration across the blood-brain barrier significantly reduces its therapeutic efficacy. To address this limitation, smart-pH nanoparticles were designed to release RIF in inflamed area and incorperated into a thermosensitive in situ gel for enhanced drug delivery via the olfactory nerve. UV-Vis spectrophotometry and colorimetric methods to quantify RIF in various media and biological matrices, including phosphate-buffered saline (PBS) at pH 7.4 and 5.0, plasma, and brain tissue. The main validation parameters assessed were selectivity, specificity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, extraction recovery, and dilution integrity. Method validation followed International Council for Harmonization (ICH) guidelines, demonstrating excellent linearity (r2 = 0.999) and lower limit detection (LOD) of approximately 0,25-0.49 μg/mL were achieved in all media. The method demonstrated high accuracy (%RE -11.62% to 14.88%) and precision (%RSD 2.06% to 13.29%), meeting regulatory requirements. This validated approach enables reliable RIF quantification in biological samples, supporting its application in in vitro release studies, ex vivo permeability studies, and in vivo pharmacokinetic evaluations. These findings contribute to the advancement of targeted drug delivery systems for TBM treatment.

A sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for the quantitation of oxymorphone (OM) in human whole blood and liver. Sample preparation was done by solid-phase extraction, using deuterated OM as the internal standard. Separation was achieved using a Waters Aquity UPLC HSS T3 column. Analysis utilized positive electrospray ionization and multiple reaction monitoring. As part of the validation, studies were conducted to determine potential interference, selectivity, ion suppression/enhancement and carryover. Calibration model, limit of detection (LOD), lower limit of quantitation (LLOQ), precision and accuracy were also established. The linear range of the method was 2-500 ng/mL in blood and 5-500 ng/g in the liver. The LOD and LLOQ were 2 ng/mL for blood and 5 ng/g for the liver. Blood and/or liver specimens from 30 cases were analyzed. OM concentrations ranged from 23 to 554 ng/mL ( , n = 26) in blood and 48 to 1740 ng/g ( , n = 30) in the liver.

A newly proposed method for detecting content of adriamycin in pectin–adriamycin conjugate has been developed and evaluated. The content of adriamycin was detected by selective degradation of adramycin to adriamycinone. It was realized by a two-phase reaction system (water–chloroform reaction system), in which adriamycin was quantitatively converted to adriamycinone. Therefore, the latter can be used to calculate the precise content of adramycin in the polymer drug. To develop the method, the catalyst for degradation, the extraction solvent for adriamycinone, the temperature and time of degradation, and the ratio of pectin–adriamycin conjugate were investigated. The optimal reaction condition was as follows: 30 mg of pectin–adriamycin conjugate dissolved in 25 mL of water was added to a mixture of 25 mL of hydrochloric acid (1.5 mol/L) and 50 mL of chloroform; the mixture was heated to 40 °C to react for 1.5 h; after that, the mixture was extracted with chloroform for three times, and then the organic layer was combined and, subsequently, evaporated to remove solvent. Under this condition, adriamycinone generation rate reached 99.87%. The quantitative method was evaluated for linearity, the limit of detection (LOD) and limit of quantitation (LOQ), recovery, accuracy, robustness, and precision. The recoveries were between 99.47% and 101.07% with relative standard deviation &lt;1.23%. The LOD and LOQ were 0.06 and 0.17 μg/mL, respectively. Compared to the traditional ultraviolet (UV) detection, this method is considered to be more precise for detecting content of adriamycin in its polymer conjugate.

The goal of this study was to investigate the extracts of ellagic acid in Korean and Chinese black raspberries according to harvest time and extraction solvent. This study was also performed to establish a simple and reliable high-performance liquid chromatography-ultraviolet (HPLC/UV) analytical method for the determination of ellagic acid for quality control and to establish baseline data of extracts of Rubus occidentalis (uRo) and Rubus chingii (uRc) fruits as healthy functional food ingredients. uRo and uRc were each extracted with 0%, 5%, 30%, and 70% ethanol. Among the extracts of these two varieties, the 5% ethanol extracts of uRo and uRc showed the highest contents of ellagic acid, 16.56 and 17.27 mg/g, respectively. The specificity was satisfied with retention time and photo-diode array (PDA) spectrum by analysis of ellagic acid using HPLC and comparison with a standard compound. Ellagic acid was validated for its limit of detection (LOD), limit of quantitation (LOQ), precision, and accuracy. It showed a high linearity in the calibration curve, with a coefficient of determination (R2) of 0.9999, and the LOD and LOQ values were 0.25 and 0.77 μg/mL, respectively. The results showed that R. occidentalis and R. chingii can be used simultaneously as healthy functional ingredients and natural medicines.

A selective, sensitive and low sample consumption GC method for determination of residual solvents in PSMA samples was developed. Selectivity with respect to solvents such as ethanol, diethyl ether, 2-propanole, acetonitrile, dichloromethane, used in manufacturing of PSMA-11, was obtained on two different GC systems and columns. In all these cases, good separation and other system suitability parameters were obtained. The values of detection limit (LOD) and limit of quantitation (LOQ) were estimated and verified experimentally for ethanol, diethyl ether, 2-propanol (IPA), acetonitrile (ACN) and dichloromethane. Full validation parameters i.e.: selectivity, limit of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy, robustness and system suitability parameters are presented with respect to ACN and IPA. Accuracy and precision were measured on two GC systems. Statistical tests exhibit, that there are no significant differences in accuracy and precision between two GC systems