Repeatability RSD Research Articles

Optimization of Microbiological Method by Turbidimetry for Quantification of Rifaximin Tablets: Validation, Application and Evaluation of Degraded Compounds

Rifaximin, an oral antibiotic, is used for the treatment of hepatic encephalopathy, ulcerative colitis, irritable bowel syndrome, Clostridium difficile, travelers' diarrhea and acute diarrhea. The development of methods which evaluate the potency of antimicrobials is fundamental. So, this work presents the validation of a microbiological method by turbidimetry to evaluate the power of rifaximin tablets, as well as the evaluation of degraded drug in acidic, basic, neutral and photolytic conditions. For this, Escherichia coli ATCC 10536 were used. The method was linear over the concentration range of 50-98 μg mL-1 with correlation coefficients 0.9976 (standard) and 0.9999 (sample), precise (repeatability RSD=4.96 and interday RSD=3.92), exact (recovery 100.70%); robust against small and deliberate variations in method and indicative of stability because it allowed evaluating rifaximin degraded under stress conditions. The results were compared with data obtained by high-performance liquid chromatography. The validated turbidimetric method is very useful to the routine quality control for microbiological evaluation of rifaximin tablets.

Evaluation of QuEChERS Sample Preparation for Determination of Avermectins Residues in Ovine Muscle by HPLC-FD and UHPLC-MS/MS

A fast and sensitive method using high performance liquid chromatography-fluorescence detection (HPLC-FD) and ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was developed and validated for the determination of avermectins residues in ovine muscle samples. QuEChERS (quick, easy, cheap, effective, rugged and safe) sample preparation based on acetonitrile extraction followed by partitioning with NaCl and Na2SO4 and dispersive solid-phase extraction (d-SPE) clean-up with C18 was applied. Na2SO4 was used instead of MgSO4 due to lower amounts of co-extractives in the final extract. The procedure was validated according to the Commission Decision 2002/657/EC. The method showed determination coefficients (r2) higher than 0.99, recoveries between 93.2 and 124.3% for spike levels between 0.5 and 2.0 times the maximum residues limit (MRL) values. The repeatability and intermediate precision RSD values ranged from 1 to 19%. Decision limits (CCα) and detection capabilities (CCβ) ranged from 10.7 to 59.4 µg kg-1 and 11.4 to 68.8 µg kg-1, respectively. Method performance was successfully evaluated by analyzing real samples and proficiency test with a z-score in the range of ±1.

Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

Development, Optimization, and Validation of a Microplate Bioassay for Relative Potency Determination of Linezolid Using a Design Space Concept, and its Measurement Uncertainty.

The aim of this study was to develop, optimize, and validate a microplate bioassay for relative potency determination of linezolid in pharmaceutical samples using quality-by-design and design space approaches. In addition, a procedure is described for estimating relative potency uncertainty based on microbiological response variability. The influence of culture media composition was studied using a factorial design and a central composite design was adopted to study the influence of inoculum proportion and triphenyltetrazolium chloride in microbial growth. The microplate bioassay was optimized regarding the responses of low, medium, and high doses of linezolid, negative and positive controls, and the slope, intercept, and correlation coefficient of dose-response curves. According to optimization results, design space ranges were established using: (a) low (1.0 μg/mL), medium (2.0 μg/mL), and high (4.0 μg/mL) doses of pharmaceutical samples and linezolid chemical reference substance; (b) Staphylococcus aureus ATCC 653 in an inoculum proportion of 10%; (c) antibiotic No. 3 culture medium pH 7.0±0.1; (d) 6 h incubation at 37.0±0.1ºC; and (e) addition of 50 μL of 0.5% (w/v) triphenyltetrazolium chloride solution. The microplate bioassay was linear (r2=0.992), specific, precise (repeatability RSD=2.3% and intermediate precision RSD=4.3%), accurate (mean recovery=101.4%), and robust. The overall measurement uncertainty was reasonable considering the increased variability inherent in microbiological response. Final uncertainty was comparable with those obtained with other microbiological assays, as well as chemical methods.

Construction and Application of Flow Enzymatic Biosensor Based of Silver Solid Amalgam Electrode for Determination of Sarcosine

AbstractIn this paper, the flow amperometric enzymatic biosensor based on polished silver solid amalgam electrode for determination of sarcosine in model sample under flow injection analysis conditions is presented. The biosensor works on principle of electrochemical detection of oxygen decrease during enzymatic reaction which is directly proportional to the concentration of sarcosine in sample. The whole preparation process takes about 3 h. The RSD of repeatability of 10 consecutive measurements is 1.6 % (csarcosine=1.0×10−4 mol dm−3). Under optimal conditions the calibration dependence was linear in the range 7.5×10−6–5.0×10−4 mol dm−3 and limit of detection was 2.0×10−6 mol dm−3.

Phytochemical analysis of Vernonanthura tweedieana and a validated UPLC-PDA method for the quantification of eriodictyol

Vernonanthura tweedieana (Baker) H. Rob., Asteraceae, is used in the Brazilian folk medicine for the treatment of respiratory diseases. In this work the phytochemical investigation of its ethanol extracts as well as the development and validation of an UPLC-PDA method for the quantification of the eriodictyol from the leaves were performed. The phytochemical study for this species lead to the identification of ethyl caffeate, naringenin and chrysoeriol in mixture, eriodictyol from leaves, and the mixture of 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-propan-1-one and evofolin B, apigenin, the mixture of caffeic and protocatechuic acid and luteolin from stems with roots, being reported for the first time for V. tweedieana, except for eriodictyol. The structural elucidation of all isolated compounds was achieved by 1H and 2D NMR spectroscopy, and in comparison with published data. An UPLC-PDA method for quantification of the eriodictyol in leaves of V. tweedieana was developed and validated for specificity, linearity, precision (repeatability and intermediate precision), limit of detection (LOD) and limit of quantification (LOQ), accuracy and robustness. In this study, an excellent linearity was obtained (r2=0.9999), good precision (repeatability RSD=2% and intermediate precision RSD=8%) and accuracy (average recovery from 98.6% to 99.7%). The content of eriodictyol in the extract of leaves of V. tweedieana was 41.40±0.13mg/g. Thus, this study allowed the optimization of a simple, fast and validated UPLC-PDA method which can be used to support the quality assessment of this herbal material.

Application of GC/MS-based metabonomic profiling in studying the therapeutic effects of Huangbai-Zhimu herb-pair (HZ) extract on streptozotocin-induced type 2 diabetes in mice.

A protocol for metabolic profiling of mice urine was developed based on gas chromatograph-mass spectrometer (GC-MS) to explore metabolic state directly. The intra-day, inter-day, repeatability, and stability RSD for most endogenous compounds were less than 3%. Type 2 diabetic mellitus (T2DM) mice model was induced by high calorie diet combined with streptozocin. Urine from the control, T2DM and Huangbai-Zhimu herb-pair (HZ) treatment mice were enrolled in the subsequent study to show the usefulness of the method. OPLS-DA scores plots demonstrate that the cluster of T2DM mice is separated from that of control mice, while HZ-T2DM mice are located close to control mice, indicating that metabolic profiles of these HZ-T2DM mice are placed toward those of control group. The results illustrate that HZ treatment could lower the level of d-glucose, hexadecanoic acid, octadecanoic acid, propanoic acid, 3-hydroxybutyric acid, and 2,3-dihydroxybutanoic acid in urine of DM mice, meanwhile the results show that HZ treatment could ameliorate T2DM symptoms by intervening the fatty acid metabolism, starch and sucrose metabolism, and glyoxylate and dicarboxylate metabolism. This preliminary application indicated that the method is suitable and reliable for urine metabolic profiling. This study might explain the metabolic effects of T2DM and the mechanisms of action of HZ against T2DM.

Simultaneous determination of multiclass preservatives including isothiazolinones and benzophenone-type UV filters in household and personal care products by micellar electrokinetic chromatography.

In this work, a simple and reliable micellar electrokinetic chromatography method for the separation and quantification of 14 preservatives, including isothiazolinones, and two benzophenone-type UV filters in household, cosmetic and personal care products was developed. The selected priority compounds are widely used as ingredients in many personal care products, and are included in the European Regulation concerning cosmetic products. The electrophoretic separation parameters were optimized by means of a modified chromatographic response function in combination with an experimental design, namely a central composite design. After optimization of experimental conditions, the BGE selected for the separation of the targets consisted of 60 mM SDS, 18 mM sodium tetraborate, pH 9.4 and 10% v/v methanol. The MEKC method was checked in terms of linearity, LODs and quantification, repeatability, intermediate precision, and accuracy, providing appropriate values (i.e. R(2) ≥ 0.992, repeatability RSD values ˂9%, and accuracy 90-115%). Applicability of the validated method was successfully assessed by quantifying preservatives and UV filters in commercial consumer products.

Development of an amperometric-based glucose biosensor to measure the glucose content of fruit.

An amperometric enzyme-electrode was introduced where glucose oxidase (GOD) was immobilized on chitosan membrane via crosslinking, and then fastened on a platinum working electrode. The immobilized enzyme showed relatively high retention activity. The activity of the immobilized enzyme was influenced by its loading, being suppressed when more than 0.6 mg enzyme was used in the immobilization. The biosensor showing the highest response to glucose utilized 0.21 ml/cm2 thick chitosan membrane. The optimum experimental conditions for the biosensors in analysing glucose dissolved in 0.1 M phosphate buffer (pH 6.0) were found to be 35°C and 0.6 V applied potential. The introduced biosensor reached a steady-state current at 60 s. The apparent Michaelis-Menten constant () of the biosensor was 14.2350 mM, and its detection limit was 0.05 mM at s/n > 3, determined experimentally. The RSD of repeatability and reproducibility of the biosensor were 2.30% and 3.70%, respectively. The biosensor was showed good stability; it retained ~36% of initial activity after two months of investigation. The performance of the biosensors was evaluated by determining the glucose content in fruit homogenates. Their accuracy was compared to that of a commercial glucose assay kit. There was no significance different between two methods, indicating the introduced biosensor is reliable.

A Rapid Method for the Determination of Vitamin D3 in Milk and Infant Formula by Liquid Chromatography/Tandem Mass Spectrometry

A rapid method for the determination of vitamin D3 applicable to milk and infant formula products is described. Samples are saponified at high temperature, and lipophilic components are extracted into isooctane in a single tube. Vitamin D3 is derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to form a Diels-Alder adduct, which is re-extracted into a small volume of acetonitrile and analyzed by UHPLC-MS/MS with quantification accomplished by an internal standard technique utilizing deuterium-labeled vitamin D3. The analysis of vitamin D3 as the PTAD adduct offers a significant increase in sensitivity and selectivity, allowing for rapid sample preparation and short chromatographic run times. The method was shown to be accurate, with spike recoveries of 94.7-104.7% and no statistical bias against both a certified reference material (P = 0.37, α = 0.05) and a reference LC-UV analytical method (P = 0.09, α = 0.05). Acceptable precision was confirmed with a repeatability RSD of 1.4-4.5% and corresponding HorRat values of 0.1-0.2. This high-throughput method is ideal for routine compliance testing, with more than 50 samples/day achievable by a single analyst.

TLC densitometric method for screening of lycopsamine in comfrey root (Symphytum officinale L.) extracts using retrorsine as a reference compound.

Due to severe toxicity of pyrrolizidine alkaloids, their quantification in medicinal products is very important. The idea of this research was to use retrorsine as a surrogate reference compound instead of lycopsamine reference or lycopsamine isolated from comfrey. A method for the analysis of lycopsamine in extracts of comfrey roots was developed and validated, employing thin layer chromatography, derivatisation with Dann-Mattocks reagent followed by densitometric analysis. The new method showed linearity within 0.70 to 7.0 μg of lycopsamine per application of 10 μL of a solution. It has also been proven to be specific and precise (repeatability RSD 2-4 % within the plate). The method was successfully employed for quantification of lycopsamine in comfrey root and comfrey root medicinal products such as ointments.

A fast and effective routine method based on HS-SPME–GC–MS/MS for the analysis of organotin compounds in biota samples

This work validated an automated, fast, and low solvent- consuming methodology suited for routine analysis of tributyltin (TBT) and degradation products (dibutyltin, DBT; monobutyltin, MBT) in biota samples. The method was based on the headspace solid-phase microextraction methodology (HS-SPME), coupled with gas chromatographic separation and tandem mass-spectrometry (GC–MS/MS). The effectiveness of the matrix-matched signal ratio external calibration was tested for quantification purposes. The exclusion of matrix influences in the calibration curves proved the suitability of this versatile quantification method. The method detection limits obtained were of 3ngSng−1dw for all the analytes. The analysis of references materials showed satisfying accuracy under optimum calibration conditions (% recovery between 87–111%; |Z-scores|<2). The repeatability RSD% and intra-laboratory reproducibility RSD% were lower than 9.6% and 12.6%, respectively. The work proved the remarkable analytical performances of the method and its high potential for routine application in monitoring organotin compounds (OTC).

SIMULTANEOUS ESTIMATION AND VALIDATION OF RAMIPRIL AND TELMISARTAN BY UV SPECTROPHOTOMETRY

A simple and sensitive spectrophotometric method for the determination of ramipril and telmisartan in pharmaceutical dosage forms has been developed. The absorption maxima were found at 220nm for ramipril and 297nm for telmisartan using 0.1N NaOH as solvent. Beer’s law was obeyed for both the drugs in the concentration range of 2-10μg/ml with correlation coefficients 0.999 for both ramipril and telmisartan. The limits of detection for ramipril and telmisartan were found to be 0.142 and 0.405μg/mL respectively and the limits of quantitation were 0.43 and 1.22μg/mL. Accuracy of the method was verified by performing recovery studies using simultaneous equation method and found to be 98.33 to 99.54%w/w for ramipril and 99.36 to 99.82 %w/w for telmisartan. %RSD of repeatability and intermediate precision studies were found to be &lt;2 for both the drugs. Ruggedness of the method was checked by changing analyst worked and instrument used. In both the cases, the %RSD was found to be less than 2.

Determination of Aloin A and Aloin B in Aloe vera Raw Materials and Finished Products by High-Performance Liquid Chromatography: Single-Laboratory Validation.

A single-laboratory validation (SLV) was conducted on an HPLC method for the detection and quantification of aloin A and aloin B in Aloe vera raw materials and finished products. An extraction procedure using sonication with an acidified solvent was used for solid test materials while liquid test materials only required dilution, if necessary, prior to filtration and analysis. Separation was achieved using a fused core C18 column in 18 min under isocratic elution conditions allowing for a single analyte (aloin A) calibration curve to quantify both aloins. Adequate chromatographic resolution (Rs ≥ 1) was achieved for aloin A and aloin B. The calibration curves for aloin A exhibited coefficients of determination (r(2)) of ≥ 99.9% over the linear range of 0.3-50 μg/mL. The LOD values were 0.092 and 0.087 μg/mL, and LOQ 0.23 and 0.21 μg/mL for aloin A and aloin B, respectively. Repeatability studies were performed on nine test materials on each of 3 separate days, with five of the test materials determined to be above the LOQ having repeatability RSD (RSDr) values ranging from 0.61 to 6.30%. Method accuracy was determined through a spike recovery study on both liquid and solid matrixes at three different levels: low, medium, and high. For both aloins, the recovery in the liquid matrix ranged from 92.7 to 106.3% with an RSDr of 0.15 to 4.30%, while for the solid matrix, the recovery ranged from 84.4 to 108.9% with an RSDr of 0.23 to 3.84%. Based on the results of the SLV study, it is recommended that this method be evaluated for reproducibility through a collaborative study.

Determination of plant stanols and plant sterols in phytosterol enriched foods with a gas chromatographic-flame ionization detection method: NMKL collaborative study.

This collaborative study with nine participating laboratories was conducted to determine the total plant sterol and/or plant stanol contents in phytosterol fortified foods with a gas chromatographic method. Four practice and 12 test samples representing mainly commercially available foodstuffs were analyzed as known replicates. Twelve samples were enriched with phytosterols, whereas four samples contained only natural contents of phytosterols. The analytical procedure consisted of two alternative approaches: hot saponification method, and acid hydrolysis treatment prior to hot saponification. As a result, sterol/stanol compositions and contents in the samples were measured. The amounts of total plant sterols and total plant stanols varying from 0.005 to 8.04 g/100 g product were statistically evaluated after outliers were eliminated. The repeatability RSD (RSDr) varied from 1.34 to 17.13%. The reproducibility RSD (RSDR) ranged from 3.03 to 17.70%, with HorRat values ranging from 0.8 to 2.1. When only phytosterol enriched food test samples are considered, the RSDr ranged from 1.48 to 6.13%, the RSD, ranged from 3.03 to 7.74%, and HorRat values ranged from 0.8 to 2.1. Based on the results of this collaborative study, the study coordinator concludes the method is fit for its purpose.

Speciation and determination of inorganic arsenic in rice using liquid chromatography-inductively coupled plasma/ mass spectrometry: collaborative study.

An analytical method to speciate two inorganic As forms [arsenite, As(lll) and arsenate, As(V)] in indica and japonica types of rice (both husked and polished) and determine the inorganic As concentration as the sum of these two was internationally validated. The method can additionally determine two organic As compounds, monomethylarsonic acid and dimethylarsinic acid, in rice as separate LC peaks. The method is based on LC separation and inductively coupled plasma (ICP)-MS detection. The method was evaluated through the International Union of Pure and Applied Chemistry/lnternational Organization for Standardization/AOAC harmonized protocol. Sixteen laboratories from four countries participated in the study, and 13 laboratories returned valid data. Twenty test portions of 10 blind duplicates of indica and japonica type rice samples (both husked and polished) were used in this study. Repeatability RSD (RSDr) and reproducibility RSD (RSDR) were calculated at five concentrations of total inorganic As between 0.03 and 0.68 mg/kg. The RSDr was in a range of 3.8 to 7.7% and the RSDR was in a range of 10 to 36%. These performance characteristics were found to be sufficient for determination of inorganic As at or higher than 0.03 mg/kg. Applicability of the method was estimated to be in a range of 0.02-2.0 mg/kg.

Interlaboratory study of immunochromatography for the rapid determination of cadmium concentrations in cereals and soybeans.

Cadmium (Cd) is one of the most toxic heavy metals to humans. To prevent the distribution of Cd-contaminated food, a simple and quick on-site test for measuring Cd concentrations in agricultural products is needed. Recently, an immunochromatography kit developed for determining Cd in rice was reported to be useful for determining Cd in many other crops. We conducted an interlaboratory study to evaluate the kit for determining Cd in cereals (wheat and rice) and soybeans. Ten test materials were used, and 12 test samples including two sets of blind duplicates were distributed to 12 laboratories in Japan. The Cd recoveries (relative to certified values or values determined by inductively coupled plasma-MS) from all test materials were 84.6-125.1%. Repeatability RSD values of the test materials ranged from 8.8 to 14.8%. Reproducibility RSD values ranged from 13.4 to 27.6%, averaging 21.3%. The Horwitz ratio ranged from 0.61 to 1.36. The reproducibility was within the range of ELISA results for measuring toxins and allergens in food. Our results indicated that the kit was an inexpensive, reliable tool for quick and easy on-site determination of Cd in cereals and soybeans.

IMEP-115: determination of methylmercury in seafood by elemental mercury analysis: collaborative study.

A collaborative study IMEP-115 was organized by the European Union Reference Laboratory for Heavy Metals in Feed and Food (EURL-HM) to validate a method for the determination of methylmercury in seafood. The method was based on a liquid-liquid extraction with an organic solvent and with an aqueous cysteine solution. The final quantitation was done with an elemental mercury analyzer. Fifteen laboratories experienced in elemental mercury analyses, from 10 European countries, took part in the exercise. Five test items were selected to cover the concentration range from 0.013 to 5.12 mg/kg. All test items were reference materials certified for the methylmercury mass fraction: DOLT-4 (dogfish liver), TORT-2 (lobster hepatopancreas), SRM 2974a (mussel), SRM 1566b (oyster), and ERM CE-464 (tuna). Participants also received a bottle of ERM CE-463 (tuna) to test their analytical method before starting the collaborative study. Method validation showed adequate accuracy and acceptable precision for all test items, thus fitting its intended analytical purpose. The repeatability RSD ranged from 3.9 to 12.3%, while the reproducibility RSD ranged from 8.4 to 24.8%.

Capillary electrophoresis of pterin derivatives responsible for the warning coloration of Heteroptera

A new capillary electrophoretic (CE) method has been developed for analysis of 10 selected derivatives of pterin that can occur in the integument (cuticle) of true bugs (Insecta: Hemiptera: Heteroptera), specifically l-sepiapterin, 7,8-dihydroxanthopterin, 6-biopterin, d-neopterin, pterin, isoxanthopterin, leucopterin, xanthopterin, erythropterin and pterin-6-carboxylic acid. Pterin derivatives are responsible for the characteristic warning coloration of some Heteroptera and other insects, signaling noxiousness or unpalatability and are used to discourage potential predators from attacking. Regression analysis defining the parameters significantly affecting CE separation was used to optimize the system (the background electrolyte (BGE) composition, pH value and applied voltage). The optimized separation conditions were as follows: BGE with composition 2mmolL−1 the disodium salt of ethylendiamintetraacetic acid, 100mmolL−1 tris(hydroxymethyl)aminomethane and 100mmolL−1 boric acid, pH 9.0, applied voltage 20kV and UV detection at 250nm. Under these conditions, all the 10 studied derivatives of pterin were baseline separated within 22min. The optimized method was validated from the viewpoint of linearity (R2≥0.9980), accuracy (relative error ≤7.90%), precision (for repeatability RSD≤6.65%), detection limit (LOD in the range 0.04–0.99μgmL−1) and limit of quantitation (LOQ in the range 0.13–3.30μgmL−1). The developed method was used for identification and determination of the contents of pterin derivatives in adults of four species of Heteroptera (Eurydema ornata cream color morph, Scantius aegyptius, Pyrrhocoris apterus and Corizus hyoscyami) and their distribution in the individual species was determined

DEVELOPMENT OF A METHOD FOR DETERMINATION OF TIOCONAZOLE ASSOCIATED WITH POLYMERIC NANOCAPSULES BY LIQUID CHROMATOGRAPHY

The aim of this work was to develop and validate an analytical method for the quantification of tioconazole in polymeric nanocapsule suspensions by high performance liquid chromatography with UV detection. The analysis was performed with a mobile phase composed of methanol:water (80:20) and 0.18% ammonium hydroxide; RP-18 column and UV detection at 219 nm. The method proved to be linear in the concentration range of 5-50 µg mL-1 (r = 0.9999), specific, precise (repeatability RSD = 1.42%, intermediate precision RSD = 1.17%), accurate (98 - 102%) and robust (RSD < 2.0%). In conclusion, a simple and rapid method was validated proving suitable for quantification of tioconazole in polymeric nanocapsules.